THE  UNIVERSITY 


OF  ILLINOIS 
LIBRARY 

^>50.1 

vn  5^ 

cl4 

C_o^>  . 2. 


Digitized  by  the  Internet  Archive 
in  2016 


https://archive.org/details/sulphurrequireme1424hart 


(?5A.rf 

\ A o A 

\A^‘ 

c^(Pv  2* 


AifllVERSiry  0F  mjtiQjm 
t AGRICULTURE  library 

g^ctUMi^ ... 

* iq 


Sulphur  Requirements  of  Farm  Crops 
in  Relation  to  the  Soil  and  Air  Supply 


BY  E.  B.  HART  AND  W.  H.  PETERSON. 

It  is  a well  known  fact  that  the  wool  of  the  sheep  is  of  a 
protein  nature.  This  protein,  keratin,  belongs  to  the  class  of 
proteins  known,  as  albuminoids  and  is  rich  in  sulphur,  contain- 
ing about  two  per  cent  in  the  air  dried  condition.  Besides  the 
sulphur  in  organic  combination  in  the  pure  wool  fiber,  there 
is  a certain  amount  of  sulphur  in  other  forms  in  the  crude 
wool. 

In  one  hundred  pounds  of  crude  wool  there  may  be,  approxi- 
mately, two  pounds  of  total  sulphur.  This  considerable  amount 
of  surphur  necessary  for  building  the  sheep’s  fleece  has  raised 
the  problem  of  the  relative  amounts  and  forms  of  this  element 
in  our  common  feeding  materials  and  the  efficiency  of  such 
forms  for  wool  production. 

Preliminary  to  the  special  investigation  on  the  relation  of  the 
form  and  supply  of  sulphur  to  wool  production  which  will  ap^ 
pear  in  future  publications,  it  was  necessary  to  determine  the 
total  sulphur  in  a number  of  our  common  feeding  materials. 
The  results  secured  led  to  a further  consideration  of  the  ques- 
tion of  the  adequacy  of  the  natural  sources  of  supply  of  this 
element  for  continuous  crop  production  and  the  data  relating 
to  that  problem  are  presented  in  this  paper. 

It  is  generally  recognized  today  by  agricultural  chemists  that 
the  amount  of  sulphur  in  plant  materials,  as  determined  in 
the  ash,  is  in  most  cases  entirely  too  low;  that  in  the  process 
of  ashing,  sulphur  is  lost  and  the  residual  amount  found  in  the 
ash  may  represent  but  a fraction  of  that  originally  present  in 
the  plant  tissue.  For  that  reason  it  is  unfortunate  that  writers 
should  speak  of  the  sulphur  in  the  ash,  so  carefully  determined 


2 


Wisconsin  Experiment  Station. 


by  Wolff1  as  representing  that  originally  contained  in  the  var- 
ious feeding  materials.  Such  losses  of  sulphur  by  ignition  have 
been  the  subject  for  study  by  a number  of  investigators.  Con- 
tributions to  this  phase  of  the  question  have  been  made  by 
Berthelot,2  Barlow,3  Fraps,4  Goss,5  Beistle,6  Sherman,7  and 
others.  The  work  of  these  several  investigators  has  served  to 
emphasize  the  inaccuracy  of  determining  the  total  sulphur  of 
plant  tissue  by  an  estimation  of  that  element  in  the  ash. 

For  the  purpose  of  our  work  a large  number  of  total  sulphur 
determinations  have  been  made  on  our  common  farm  products. 
For  this  work  the  peroxide  method,  as  outlined  by  Osborne8  has 
been  used.  The  modification  of  this  method,  as  given  in  the 
Official  Methods  of  the  Association  of  Agricultural  Chemists,9 
was  used  for  duplicate  determinations  in  a number  of  cases. 
Since  these  determinations  were  in  no  closer  agreement  than 
duplicates  by  the  Osborne  method,  the  latter  was  preferred. 
The  addition  of  sodium  carbonate  in  the  official  method  makes 
the  oxidation  slower,  the  fusion  harder  to  remove  from  the 
crucible  and,  unless  care  is  taken  to  have  the  sample  well 
moistened,  flashing  and  burning  are  very  liable  to  occur  when 
the  peroxide  is  added. 

Instead  of  completely  boiling  off  the  water  from  the  hydroxide, 
as  directed  in  the  Osborne  method,  a little  should  be  left.  It 
helps  to  prevent  frothing  when  the  sample  is  added  and  secures 
a more  complete  saturation  of  the  material  with  the  hydroxide. 
No  hydrochloric  acid  was  used  in  removing  the  fusion  from 
the  crucible  as  it  was  found  that  hot  water  would  do  this  com 
pletely.  The  black  coating  on  the  inside  of  the  crucible  is  thus 
retained  and  the  life  of  the  crucible  measurably  prolonged. 

Amounts  of  Sulphur  Trioxide  in  Feeding  Materials. 

The  results  secured  by  the  Osborne  method  are  reported  in 
Table  I.  All  determinations  were  made  on  the  air  dried  ma- 
terials. For  convenience  of  reference  the  results  are  reported 
both  as  elemental  sulphur  and  as  sulphur  trioxide,  although  in 


1 Wolff’s  Aschen  Analysen. 

2 Compt.  Rend.  128;  17. 

s Jour.  Amer.  Chem.  Soc.,  1904,  26:  341. 

4 N.  C.  Expt.  Sta.  Ann.  Rept.,  1901-3. 

s N.  Mex.  Expt.  Sta.  Bui.,  44 

e Jour.  Amer.  Chem.  Soc.,  24:  1093. 

7 Jour.  Amer.  Chem.  Soc.,  24:  1100. 

8 Jour.  Amer.  Chem.  Soc.,  24:  142. 

9 U.  S.  Dept.  Agr.  Bur.  Chem.  Bui.  107 . 


Sulphur  Requirements  of  Farm  Crops 


3 


our  discussion  of  the  subject  the  latter  formula  for  expressing 
the  amount  of  sulphur  will  be  used.  In  addition  to  the  total 
amount  of  sulphur  trioxide  reported  there  is  given  in  a num- 
ber of  cases  the  amount  contained  in  the  ash  as  secured  oy 
Wolff. 


Table  I.— Sulphur  in  Feeding  Stuffs — Air  Dry. 


Material.  ' 

Sulphur. 

| Sulphur 
| Trioxide. 

Sulphur 
Trioxide 
| in  ash.* 

Per  cent 

Per  cent 

Per  cent 

Alfalfa  hay 

0.287 

0.717 

0.42-5 

0.292 

0.730 

Bariev  

0.153 

0.E82 

0.060 

Barley  straw  

0.147 

0,367 

0.207 

Beans  (white  garden) 

0.232 

0,580 

0.130 

0.136 

0.341 

Cabbage  

0.819 

2.047 

1.317 

Clover,  red  

| 0.164 

0.410 

0.222 

Corn,  white  

0.170 

0.425, 

0.010 

0.139 

0.347 

0.139 

0.347 

Corn  stover,  sample  1 

0.126 

0.315 

0.282 

0.116 

0.290 

0.115 

0.287 

0.128 

0.320 

<~!nrn  silagp  

0.122 

0.305 

Cotton  seed  meal 

0.487 

1.217 

0.092 

Flnnr  whpat,  

0.180 

0.450 

Flnnr,  graham  

0.183 

0.457 

Glntpn  fppd,  sample  1 

0.560 

1.399 

Glnten  feed,  sample  9.  

0.5C0 

1.249 

Hay,  mixed  

0.160 

0.400 

0.354 

Linseed  meal,  sample  1 

0.404 

1.010 

0.190 

Linsppd  meal,  sample  2’ 

0.375 

0.937 

Oats,  sample  1 

0.189 

0.472 

0.055 

Oats,  sample  2 

0.180 

0.450 

Oatmpal.  sample  1 

0.223 

0.557 

Oatmeal,  sample  2 

0.228 

0.570 

Oat  straw,  sample  1 

0.195 

0.487 

0.230 

Oat  straw,  sample  2 

0.218 

0.545 

Onions  

0.568 

1.419 

0.300 

Potatoes  

0.137 

0.343 

Rape  tops  

0.988 

2.470 

| 1.132 

Rapeseed  meal  

0.456 

1.140 

0.381 

Rice  

0.126 

0.315 

0.003 

Rice  bran  

0.181  | 

0.452  1 

0.0.22 

Rutabagas,  sample  1 

0.817 

2.041 

| 

Rutabagas,  samnle  2 

0.632 

1.579 

Rye  

0.123 

; 0.309 

| 

Rye  straw  

0.049 

0.123 

Soy  bean  

0.341 

0.852 

0.085 

Sugar  beet,  late  field  sample 

0.128 

0.320 

0.160 

Sugar  beet,  stored  roots  

0.089 

0.222 

Sugar  beet,  early  field  sample 

0.039 

0.172 

Sugar  beet  tops 

0.433 

1.082 

0.790 

Timothy  

0.190 

0.475 

0.195 

Turnips  

0.740 

1.849 

0 897 

Turnip  tons  

0.900 

2.249 

1.095 

Wheat,  sample  1 

0.170 

0.425 

0.007 

Wheat,  sample  2 

0.164 

0.410 

Wheat,  sample  3 

0.176 

0.440 

Wheat  bran,  sample  1 

0.200 

0.499 

0.005 

Wheat  bran,  sample  2 

0.224 

0.559 

Wheat  gluten  

0.860 

2.149 

0.011 

Wheat  straw,  sample  1 

0.119 

0.297 

0.132 

Wheat  straw,  sample  2 

0.160 

0.399 

[ — i 

Wolff’s  Aschen  Analysen. 


4 


Wisconsin  Experiment  Station 


Table  I discloses  some  very  interesting  facts.  The  results 
are  in  harmony  with  those  of  other  investigators  in  showing 
a much  higher  content  of  total  sulphur  trioxide  in  plant  ma- 
terials than  is  found  in  the  ash.  This  is  particularly  true  in  the 
seeds  where  the  sulphur  exists  largely  in  organic  form  in  the 
protein  molecule.  There  the  loss  by  ignition  is  very  large. 

There  is  one  hundred  times  as  much  sulphur  trioxide  in  rice 
grain  as  in  the  ash  of  that  grain  and  forty  times  as  much  in  the 
corn  grain  as  in  its  ash.  In  wheat  the  same  condition  is  true,  while 
in  the  oat,  cottonseed  and  soy  bean  the  total  sulphur  trioxide 
recovered  in  the  grain  by  the  fusion  method,  is  about  ten  times 
that  found  in  the  ash.  In  onions,  cabbage  and  rutabagas  the 
amount  found  in  the  green  tissue  is  from  two  to  four  times 
that  recovered  in  the  ash.  These  materials  contain  volatile  sul- 
phur oils  which  are  in  part  responsible  for  the  sulphur  lost  on 
ignition. 

In  the  case  of  the  hays  and  straws  the  losses  of  sulphur  by 
ignition  have  not  been  so  large  as  in  the  seeds  and  in  those 
plants  rich  in  sulphur  oils.  Presumably  a considerable  part  of 
the  sulphur  exists  in  plant  stems  as  sulphates  and  consequently 
is  permanent  on  ignition.  In  mixed  hay,  for  example,  the  amount 
of  sulphur  trioxide  recovered  in  the  ash  is  nearly  as  large  as 
that  found  in  the  original  material,  but  in  alfalfa  and  clover 
hay  the  recovery  amounts  to  about  50  per  cent.  Of  course  the 
probable  variation  in  the  total  sulphur  content  of  different  sam- 
ples of  material  must  be  taken  into  consideration,  as  the  sulphur 
content  of  the  ash  is  here  quoted  from  Wolff’s  analyses.  This 
would  apply  especially  to  such  parts  of  the  plant  as  the  stem 
and  leaf,  but  not  in  the  same  degree  to  the  seed the  data  serve 
to  emphasize  the  general  truth  of  the  large  loss  of  sulphur  on 
ignition  from  seeds  and  considerable  loss  from  the  tissues  of 
members  of  the  Cruciferae  and  a somewhat  smaller  amount  of 
loss  from  the  hays  and  straws. 

Amounts  of  Sulphur  Trioxide  Removed  by  Crops. 

The  very  important  fact  which  the  above  data  furnishes  is 
that  farm  crops  remove  much  more  sulphur  from  the  soil  than 
has  been  supposed.  Based  on  Wolff’s  ash  analyses  a 100  bushel 
corn  crop  per  acre  (grain  only)  would  remove  about  one-half 
of  a pound  of  sulphur  trioxide,  while  the  actual  total  sulphur 


• Sulphur  Requirements  of  Farm  Crops 


o 


trioxide  removed,  according  to  our  analysis,  would  be  over  20 
pounds.  For  the  purpose  of  clearly  showing  what  amounts  of 
sulphur  trioxide  are  removed  by  average  farm  crops  Table  II 
has  been  constructed. 


Table  II — Pounds  of  Sulphur  Trtoxide  and  Phosphorus  Pentoxide 
Removed  Per  Acre  by  Average  Crops. 


Crop.  A 

1 

j Dry 

1 weight. 

1 

1 From  Wolff’s 
ash  analyses 
sulphur 
trioxide. 

Actual 

sulphur 

trioxide. 

Phosphorus 

pentoxide. 

Pounds 

Pounds 

Pounds 

Pounds 

Wheat  grain,  30  bushels 

1,530 

0.15 

6.4 

14.2 

Wheat  straw 

2,653 

3.40* 

9.3 

6.9 

Total  crop  

4,183 

3.55 

15.7 

1 

21.1 

Barley,  grain,  40  bushels 

1,747 

1.0 

6.6 

16.0 

Barley  straw  

1 2,080 

j 4.1 

7.7 

4.7 

Total  crop  

3, 827 

5.1 

14.3 

20.7 

Oat  grain,  45  bushels 

1,625 

0.8 

7.5 

13.0 

Oat  straw  

2,353 

5.4 

12.2 

6.4 

Total  crop  

3,978 

6.2 

19.7 

19.4 

Corn  grain,  30  bushels 

1,500 

0.15 

6.4 

10.0 

Corn  stalk  

1,877 

5.20 

5.6 

8.0 

Total  crop  

3,377 

5.35 

12.0 

18.0 

Meadow  hay  

2,822 

9.8 

11.3 

12.3 

Red  clover  hay 

3,763 

8.2 

15.4 

24.9 

Alfalfa  hay  

9,000 

37.8 

64.8 

39!  9 

Bean  grain,  30  bushels 

1,613 

9 4 

<>•7  Q 

Bean  straw  

1,848 

4.9 

6.3 

Total  crop  

3,461 

14.3 

29.1 

Turnips,  root  

3,126 

27.8 

57.8 

•>?  4 

Turnips,  leaf  

1,531 

16.6 

34.4 

l6l7 

Total  crop  

4,657 

44.4 

92.2 

33.1 

Sugar  beet  root 

4,320 

6.9 

9.5 

90  91 

Sugar  beet  leaf 

1,848 

14.5 

20.0 

13.1 

Total  crop  

6,i68 

21.4 

29.5 

33.3 

Potatoes  

3,360 

1 1 CTO1 

Tobacco  leaf  

1,800 

11. 5Z 

1 PL 

21.5 

Tobacco  stalk  

S’ 200 

lo. 

8. 



5. 

8. 

Total  crop  

5,000 

1 

Cabbage  

4,800  1 

62.8  | 

Zl. 

98. 

16. 

61. 

For  purposes  of  comparison,  the  amounts  removed  as  calcu- 
lated from  Wolff’s  ash  analyses  are  given  in  a number  of  cases. 
In  addition  to  the  above  figures  there  are  also  included  the 
amounts  of  phosphorus  pentoxide  removed  by  average  crops. 
This  is  done  for  the  purpose  of  comparing  the  amounts  of  these 


6 Wisconsin  Experiment  Station 

two  very  essential  constituents  removed  by  plant  growth.  It 
will  also  serve  as  a basis  for  the  proper  treatment  of  the  ques- 
tion of  sulphur  fertilization. 

A study  of  Table  II  reveals  the  fact  that  the  average  crop  of 
seed  from  the  cereal  plants  removes  from  the  soil  about  half  as 
much  sulphur  trioxide  as  phosphorus  pentoxide  and  that  the 
straws  remove  a somewhat  larger  quantity.  The  hays  are  not 
widely  different  in  the  proportion  of  these  important  ingredients 
removed,  although  alfalfa  removes  annually  twice  as  much  sul- 
phur trioxide  as  phosphorus  pentoxide.  The  Cruciferae  are 
heavy  sulphur-using  plants  and  the  total  sulphur  trioxide  re- 
moved by  these  is  large.  An  average  acre  crop  of  turnips  or 
cabbage  appropriates  nearly  100  pounds  of  this  compound. 

Amounts  of  Sulphur  Trioxide  in  Soils. 

No  one  questions  the  absolute  necessity  of  sulphur  for  plant 
growth.  It  is  necessary  for  the  production  of  plant  proteins 
and  all  the  plant  proteins  that  have  been  investigated  contain 
sulphur.  Only  one  class  of  proteins  is  known  to  be  free  from 
sulphur  and  that  is  the  class  of  protamines  of  animal  origin 
which,  however,  have  not  as  yet  been  isolated  from  plant  tissue. 

The  imperative  necessity  of  maintaining  an  ample  supply  of 
this  element  for  plant  production  is  as  important  as  maintain- 
ing a supply  of  phosphorus,  nitrogen  or  any  of  the  other  ele- 
ments essential  for  plant  development.  The  apparent  reason 
why  so  little  attention  has  been  given  to  this  element  in  the 
schemes  of  fertilization  for  plant  production  has  been  due  to  the 
fact  that  it  was  believed  that  crops  removed  but  little  from  the 
soil  and  consequently  the  supply  was  ample  for  continuous  pro- 
duction. 

Bogdanov10  in  1899  called  attention  to  .the  practical  impor- 
tance of  sulphur  in  agriculture  and  believed  that'  it  should 
be  applied  occasionally  as  a sulphate  for  the  express  purpose  of 
maintaining  in  the  soil  an  adequate  supply  of  this  element. 
Dymond.  Hughes  and  Dupe,* 11  have  also  touched  upon  this  sub- 
ject and  concluded  that  there  was  not  sufficient  sulphur  in  the 
soil  for  the  greatest  yield  of  crops  rich  in  protein,  but  that  for 
cereal  crops  and  permanent  pasture  the  soil  and  rain  would  pro- 


10  Abstract  Expt.  Sta.  Rec.,  11:  723. 

11  Jour,  of  Agr.  Sci.,  1905,  1:  217. 


Sulphur  Requirements  of  Farm  Crops 


vide  a sufficient  quantity.  So  far  as  we  can  find  in  the  litera- 
ture no  further  treatment  of  the  subject  has  been  made. 

Investigation  of  the  forms  of  sulphur  in  soils  has  been  made 
by  Berthelot  and  Andre.12  According  to  these  investigators  sul- 
phur exists  in  soil  as  (1)  sulphates  and  sulphides;  (2)  ethereal 
sulphur;  (3)  in  organic  compounds.  Very  probably  the  chief 
forms  in  all  normal  soils  will  be  those  existing  as  sulphates  and 
those  in  the  organic  matter  of  the  soil.  Sulphides  would  rarely 
exist  except  in  waterlogged  soils  where  reducing  processes  are 
prominent.  Ethereal  sulphates  would  probably  be  found  only 
after  fertilization  with  the  urine  of  animals,  and  then  only  in 
small  amounts. 

There  are  some  difficulties  in  the  estimation  of  the  total  sul- 
phur of  soils.  Fusion  of  the  soil  itself  with  an  alkali  is  some- 
what tedious,  although  probably  the  most  accurate  procedure. 
The  large  amount  of  silicates  and  their  incomplete  removal  be- 
fore final  precipitation  with  barium  chloride,  may  endanger  the 
absolute  accuracy  of  the  method.  Extraction  in  the  wet  wa^ 
with  strong  oxidizing  agents,  while  probably  not  absolute,  will 
nevertheless  give  the  amount  of  sulphur  that  can  reasonably  be 
expected  to  become  available  to  the  crop  in  future  years.  Van 
Bemmelen13  compared  several  methods  for  the  estimation  of  sul- 
phur in  soils,  obtaining  the  highest  results  by  extraction  with 
aqua  regia.  Trials  by  the  method  of  ignition  with  sodium  car- 
bonate and  potassium  nitrate  and  the  method  of  ignition  in  a 
stream  of  oxygen  as  used  by  Berthelot  gave  him  slightly  lower 
results  than  with  aqua  regia.  It  is  safe  to  assume  that  diges- 
tion and  extraction  with  a strong  oxidizing  agent  will  remove 
all  sulphates,  oxidize  sulphides  to  sulphates,  and  split  up  any 
ethereal  sulphates  present  to  form  sulphuric  acid,  and  at  least 
partly  oxidize  the  sulphur  existing  in  organic  forms.  It  prob- 
ably will  not  give  the  total  sulphur  present  in  the  organic  ma- 
terial of  the  soil. 

Most  of  the  determinations  of  sulphur  in  soils  carried  out 
heretofore  have  been  made  by  extraction  with  strong  hydro- 
chloric acid.  This  has  been  done  either  by  the  long  time  ex- 
traction method  as  recommended  by  Hilgard  or  by  the  method 
adopted  by  the  Association  of  Official  Agricultural  Chemists. 


12  Ann.  Chem.  et  Phys.,  1892,  25:  305. 
is  Land.  Vers.  Stat.,  1890,  37:  284. 


8 


AVisconsin  Experiment  Station 


These  methods  will  presumably  give  all  sulphates  and  ethereal 
sulphur,  but  not  that  existing  as  sulphides,  or  the  sulphur  in 
combination  with  the  organic  matter  of  the  soil.  Hilgard  found 
from  his  analyses  of  many  types  of  soil,  that  the  average  amount 
of  sulphur  trioxide  in  sandy  soils  was  0.055  per  cent  wdiile  in 
the  clay  soils  examined,  it  amounted  to  0.075  per  cent.  This,  on 
the  basis  of  an  acre  foot  of  three  million  pounds,  would  amount 
in  the  first  instance  to  1,650  pounds,  and  in  the  latter  case  to 
2,250  pounds.  The  average  amount  of  phosphorus  pentoxide  as 
given  by  Hilgard  for  the  same  sandy  soils  was  0.087  per  cent 
and  0.141  per  cent  for  the  clay  soils.  On  the  same  basis  of  cal- 
culation an  acre  foot  would  contain  in  the  first  case  2,610 
pounds  and  in  the  second  4,230  pounds  of  phosphorus  pentoxide. 

AVhitson14  and  Stoddart  also  have  shown  that  the  average 
phosphorus  pentoxide  contents  for  the  surface  eight  inches  of 
many  AVisconsin  soils  is  about  2,940  pounds. 

These  figures  are  introduced  for  the  purpose  of  showing  that 
the  amount  of  sulphur  in  all  normal  soils  is  comparatively  low. 
Of  course  the  quantity  would  not  be  considered  low  if  the 
amount  removed  by  crops  was  also  relatively  small;  but  com- 
pared with  phosphorus  the  amount  of  sulphur  in  normal  soils 
is  on  the  average  percentagely  lower,  while  the  amount  of  sul- 
phur removed  by  crops  is,  relative  to  the  supply,  quite  as  large. 
In  some  cases,  at  least,  as  with  the  turnip,  cabbage  and  other 
plants  of  the  Cruciferae  family,  the  amount  of  sulphur  removed 
is  very  much  greater  than  the  amount  of  phosphorus  removed. 

Effect  of  Continuous  Cropping  on  the  Sulphur  Content  of 

the  Soil. 

To  determine  definitely  the  effect  of  continuous  cropping  on 
the  sulphur  content  of  soils,  a number  of  analyses  of  both 
cropped,  virgin  and  manured  soils  have  been  made.  Two 
methods  were  used  for  the  determination  of  sulphur  trioxide. 

The  first  was  a slight  modification  of  the  Yan  Bemmelen 
method.  That  investigator  used  aqua  regia  for  the  extraction. 
Our  modification  was  as  follows : — 10  grams  of  the  air-dried  soil 
were  placed  in  a flask,  fitted  with  a ground  glass  stopper, 
in  which  was  fused  a glass  tube  about  two  feet'  long  to  serve  as 


14  Wis.  Expt.  Sta.  Res.  Bui.  2,  1909,  p.  42. 


Sulphur  Requirements  of  Farm  Crops  9 

a return  condenser.  Two  to  three  cc.  of  bromine  were  added  to 
the  soil  and  then  50  cc.  of  strong  nitric  acid,  sp.  gr.  1.42;  the 
flask  and  contents  were  then  thoroughly  shaken  and  finally 
placed  on  the  steam  bath  and  digested  for  24  hours.  Bromine 
was  added  occasionally  to  replace  that  lost  by  evaporation.  The 
whole  operation  was,  of  course,  carried  on  in  a well  ventilated 
hood.  After  digesting  24  hours  the  flask  was  cooled,  fil- 
tered and  the  residue  thoroughly  washed  with  hot  w^ater  until 
at  least  a volume  of A 200  cc.  had  been  collected.  The  clear  fil- 
trate was  then  evaporated  to  dryness  and  the  residue  treated 
with  hydrochloric  acid.  This  solution  w7as  again  evaporated  to 
dryness  and  the  operation  with  hydrochloric  acid  repeated  twice 
in  order  to  insure  complete  removal  of  all  nitric  acid.  The 
residue  was  then  taken  up  with  water  slightly  acidulated  with 
hydrochloric  acid,  digested  hot  for  half  an  hour,  filtered  and 
thoroughly  washed.  The  solution  was  neutralized  with  am- 
monia, and  then  made  acid  with  4 cc.  of  hydrochloric  acid 
brought  to  boiling  and  precipitated  with  barium  chloride.  Af- 
ter standing  w^arm  for  24  hours  the  precipitate  was  filtered, 
ignited  and  weighed. 

The  second  method  consisted  of  fusion  with  sodium  peroxide. 
Ten  grams  of  soil  wrere  placed  in  a 100  cc.  nickel  crucible,  moist- 
ened with  water,  about  10  grams  of  a weighed  20  gram  portion 
of  sodium  peroxide  added,  and  the  mixture  thoroughly  stirred 
with  a platinum  rod.  The  crucible  was  placed  over  an  alcohol 
flame  and  heated  moderately  until  the  mass  was  dry.  The  re- 
mainder of  the  sodium  peroxide  was  then  added,  the  cover 
placed  on  the  crucible,  strong  heat  applied  until  the  mass  melted, 
and  kept  in  this  condition  for  10  minutes.  It  was  then  allowed 
to  stand  over  a lower  flame  for  1 hour.  The  crucible  was  re- 
moved, cooled,  placed  in  a 600  cc.  casserole,  hot  water  added 
and  the  fused  mass  removed.  It  was  neutralized  with  hydro- 
chloric acid  and  then  further  acidified  with  10  cc  of  hydro- 
chloric acid.  The  volume  was  made  up  to  about  450  cc.  and 
boiled  for  15  minutes,  or  until  no  undecomposed  portion  of  the 
fused  mass  remained  on  the  bottom.  The  covered  casserole  w^as 
allowed  to  stand  on  the  steam  bath  over  night,  filtered  through 
a ‘ ‘ nutsche  ’ ’ and  the  residue  thoroughly  wrashed  with  successive 
small  portions  of  hot  water.  The  filtrate  and  washings,  if  over 
500  cc.,  were  evaporated  below  that  volume,  refiltered  and  the 


10 


Wisconsin  Experiment  Station 


volume  made  up  to  500  cc.  Aliquots  of  250  cc.  each  were 
heated  to  boiling,  barium  chloride  added,  boiled  for  5 minutes 
and  set  aside  on  a steam  bath  for  24  hours.  The  volume  was 
not  allowed  to  decrease  as  silicic  acid  may  be  precipitated 
if  much  evaporation  takes  place.  After  standing  for  this 
length  of  time  the  barium  sulphate  was  filtered  off,  washed,  ig- 
nited and  weighed.  In  the  determinations  made  by  this  method 
the  precipitate  was  free  from  silica  as  demonstrated  by  the 
hydrofluoric  acid  test. 

Both  these  methods  were  compared  with  the  method  of  the 
Official  Agricultural  Chemists  with  the  following  results : The 

soil  used  was  from  the  surface  8 inches  of  one  of  the  experi- 
mental plots  at  the  University  Hill  Farm. 

Per  cent  of  SOr 


Official  method  0.019 

Fusion  method  043 

Nitric  acid-bromine  method 037 


Since  the  official  method  gave  lower  results  than  either  the 
fusion  or  wet  extraction  method  it  was  not  used  for  our  work. 
Instead,  for  the  purpose  of  greater  completeness,  all  the  soils 
investigated  were  subjected  to  both  the  fusion  and  nitric  acid- 
bromine  methods.  There  was  close  agreement  between  the  re- 
sults secured  by  the  two  methods,  but  with  a uniform  tendency 
for  the  fusion  method  to  give  slightly  higher  results.  This  is 
to  be  expected  as  that  method  should  include  the  total  sulphur 
of  the  soil,  while  by  the  wet  method  some  of  the  organic  sulphur 
may  escape  complete  oxidation.  In  the  analyses  reported  only 
those  results  secured  by  the  fusion  method  are  recorded. 

Sulphur  Trioxide  in  Soil  Samples. 

Ten  different  soils  from  several  parts  of  this  state  were  first 
investigated.  They  were  both  virgin  and  the  same  soils,  cropped 
but  generally  unmanured.  Some  of  these  were  kindly  furnished 
us  by  the  Soils  Department  of  this  Station  and  had  already  been 
the  subject  for  investigation  by  that  department  of  losses  in 
phosphorus.  The  determinations  of  sulphur  trioxide  were  made 
on  pairs  of  samples,  one  from  the  cropped  field  and  the  other 
from  the  adjacent  virgin  soil.  In  each  case  the  surface  8 inches 
were  taken  and  every  precaution  to  secure  virgin  soils  of  drain- 


Sulphur  Requirements  of  Farm  Crops 


11 


age  and  topography  similar  to  that  of  the  cropped  soil  was  ob- 
served. Analyses  are  also  given  in  the  later  part  of  this  article 
of  virgin  soils  and  the  same  soils  heavily  manured,  but  in  two 
instances  continuously  cropped  to  such  heavy  sulphur  using 
plants  as  the  cabbage. 

The  following  is  a brief  history  of  the  unmanured  samples. 
Samples  1,  2 and  3 furnished  by  the  Soils  Department,  were 
identical  with  those  described  in  Research  Bulletin  No.  215  of 
this  Station,  page  44, ,s  under  numbers  7,  8 and  9,  respectively. 

No.  1 (Cropped).'  “Janesville.  Cropped  63  years.  Dur- 
ing the  first  34  years  wheat  was  grown  almost  continuously. 
Since  1878  it  has  been  rotated  to  corn,  barley,  oats  and  rye.  It 
has  never  been  seeded  down  or  manured  and  is  in  a badly  ex- 
hausted condition.” 

No.  2 (Cropped).  “Edgerton.  Cropped  about  60  years, 
largely  to  wheat  at  first,  but  during  the  last  40  years  it  has  been 
farmed  in  a rotation,  consisting  of  two  crops  of  corn,  one  of 
oats  and  two  of  clover  and  timothy,  of  which  the  first  was  cut 
and  the  second  pastured.  The  field  has  not  produced  good  crops 
during  the  last  10  years.  It  has  been  manured  but  once.” 

No.  3 (Cropped).  “Milton  Jet.  Cropped  52  years,  chiefly 
to  wheat  during  the  first  15  years,  since  then  it  has  been  rotated 
to  oats  and  barley  with  a few  years  of  timothy.  It  has  been 
manured  10  times  with  an  average  of  10  loads  per  acre  each 
time.  The  field  dees  not  now  produce  good  crops.” 

No.  4 (Cropped).  “Milton  Jet.  Cropped  50  years.  Rotated 
to  corn  and  oats  for  about  40  years,  then  grew  one  crop  of  bar- 
ley, one  of  corn,  one  of  beets  and  now  in  alfalfa.  Alfalfa  doing 
poorly.  Soil  acid.  Manured  once.” 

No.  5 (Cropped).  “Evansville.  Cropped  60  years,  early 
years  chiefly  in  wheat.  History  for  last  28  years — 10  years 
timothy  and  clover ; 12  crops  of  corn ; 6 crops  of  oats.  Manured 
three  times.  Manure  usually  sold.” 

Table  No.  3 gives  the  results  of  the  analyses  of  the  virgin  and 
cropped  soils ; the  loss  of  sulphur  trioxide  by  cropping  as  deter- 
mined from  the  analyses;  the  estimated  amount  of  sulphur  tri- 
oxide removed  by  the  crops;  the  amount  added  in  the  manure; 
and  the  amount  removed  by  the  crop  in  excess  of  that  added  in 


Whitson,  A.  R.  and  Stoddart,  C.  W.  “Factors  Influencing  the  phos- 
phate content  of  soils.”  Wis.  Expt.  Sta.  Research  Bui.  2,  1909,  p.  44. 


12 


Wisconsin  Experiment  Station 


the  manure.  It  is  estimated  that  the  average  amount  of  sul- 
phur trioxide  removed  every  year  by  corn,  the  small  grains, 
clover  and  timothy  was  12  pounds.  This  amount  is  somewhat 
below  the  figure  given  in  Table  II  for  average  crops,  but  the 
smaller  amount  is  taken  because  of  the  falling  off  of  crop  yields 
in  the  later  years  of  the  field’s  history.  The  average  amount 
of  sulphur  trioxide  added  in  the  manure  was  estimated  on  the 
basis  of  several  analyses,  at  two  pounds  per  ton  of  manure  with 
an  application  of  10  tons  yearly. 

Table  III  indicates  that  on  the  average  about  40  per  cent  of 
the  sulphur  trioxide  has  been  lost  from  the  cropped  soils.  In 
every  case  there  is  a lower  percentage  of  sulphur  trioxide  in 
the  cropped  soil  than  in  the  virgin  soil.  The  estimated  amount 
removed  by  the  crops,  minus  that  supplied  in  the  manure,  is  in 
two  cases  in  excess  of  that  indicated  to  have  been  lost  by  the 
analysis.  Such  discrepancies  are  due,  probably,  to  variations 
in  the  soil  itself,  inaccurate  estimates  of  the  amounts  of  crop 
removed,  and  the  influence  of  the  influx  of  sulphates  with  the 
upward  movement  of  soil  water.  On  the  other  hand  the  esti- 
mated average  amounts  of  sulphur  trioxide  removed  by  the 
crops  and  tflat  indicated  by  the  analyses,  are  almost  identical. 
This  estimate  cn  the  soil  is  based  on  the  surface  8 inches  and 
the  weight  of  two  million  pounds  per  acre.  Little  importance, 
however,  should  be  attached  to  these  estimated  amounts  removed, 
as  there  must  still  be  losses  of  sulphur  trioxide  due  to  drainage 
which  the  figures  do  not  include.  The  important  thing  that  the 
table  teaches  is,  that  continuous  cropping  without  adequate  fer- 


Table  III. — Influence  of  Exhaustive  Cropping  on  the  Sulphur 
Trioxioe  Content  of  Soil. 


Soil 

sample 

No. 

! 

SOn  in 
virgin 
s.  il. 

SO  a in 
cropped 
soil. 

Amount  Per  Acre  (Estimated). 

Loss  by  crop 
ping'arcot-ding- 
to  analyses. 

Removed 
by  crops. 

Added  in 
manure. 

Remo  ed  by 
crop  in  excess 
of  that 
added  in 
manure. 

Per  cent. 

Percent. 

Pounds. 

Pounds. 

Pounds. 

Pounds. 

1 

0 107 

| 0 . 0fi6 

8?0 

756 

7^6 

0.101 

0*058 

860 

720 

20 

700 

3 

0.033 

0.010 

280 

624 

200 

424 

4 

0.127 

0.088 

780 

600 

20 

580 

0.055 

0.032 

460 

720 

1,0 

660 

Average. 

0.084 

0.052 

640 

684 

60 

624 

Sulphur  Requirements  of  Farm  Crops 


13 


tilization  shows  a large  decrease  in  the  snlphnr  trioxide  content 
of  the  soils  examined. 

Effect  of  Liberal  Manuring  on  the  Sulphur  Trioxide  Con- 
tent of  Soils. 

In  order  to  determine  what  would  be  the  effect  of  liberal 
manuring  with  farm  manure  on  the  sulphur  trioxide  content 
of  soils,  several  samples  of  known  history  were  subjected  to  an 
examination.  The  corresponding  virgin  soils  were  also  exam- 
ined. The  history  of  the  cropped  samples  is  as  follows: 

1 No.  6.  “Evansville.  Cropped  60  years.  Wheat  up  to  1860. 
Since  1860  general  rotation  of  two  years  in  corn,  one  to  two 
years  in  meadow,  and  one  year  in  pasture.  Manured  every  year 
' while  in  corn  at  the  rate  of  10  loads  to  the  acre.  Crop  yields 
good. ? ’ 

No.  7.  “Berryville.  Continuously  in  cabbage  until  the  crop 
failed  some  10  years  ago.  Manured  heavily  with  stable  manure 
until  three  years  ago.  The  last  three  years  fertilized  annually 
with  1,100  pounds  per  acre  of  Homestead  fertilizer.  Surface 
two  inches  rejected  in  taking  samples.” 

No.  8.  “Kenosha.  Past  15  years  in  cabbage,  alternating 
every  year  with  corn  or  potatoes.  Chicago  stable  manure  freely 
applied  with  yearly  applications  of  about  10  loads.  None  used 
in  1909.” 

The  results  are  given  in  Table  IV. 


Table  IV. — Influence  of  Liberal  Manuring  With  Farm  Manure  on 
the  Sulphur  Trioxide  Content  of  Soils. 


Soil  sample  No. 

SO s in 
virgin  soil. 

SO  :>  in  cropped  and 
manured  soil. 

6 

Per  cent. 
0.061 
.103 

.110 

Per  cent. 
0.075 
.05 
.140 

7 

>8  

Average 

.096 

.110 

The  results  show  that  the  sulphur  content  of  these  soils  was 
maintained  and  even  slightly  increased  by  the  liberal  applica- 
tion of  farm  manure. 


14 


Wisconsin  Experiment  Station 


Other  Factors  in  the  Gain  and  Loss  of  Sulpher  Trioxide 

from  Soils. 

In  addition  to  the  sulphur  added  to  the  soil  with  applications- 
of  farm  manure  or  certain  commercial  fertilizers  there  is  also 
a small  amount  brought  to  the  land  by  rain  water.  This  sul- 
phur in  the  atmosphere  has  its  origin  almost  wholly  in  the  burn- 
ing of  fuel,  especially  soft  coal.  Where  soft  coal  is  abundantly 
consumed  the  amount  may  be  expected  to  be  somewhat  larger 
than  where  anthracite  coal  is  the  chief  fuel  used.  The  sulphur 
exists  in  the  atmosphere  as  sulphur  dioxide  and  trioxide  and  is 
brought  down  in  the  rain  either  as  the  free  acids  or  salts  of 
these  acids.  It  is  very  probable  that  part  of  the  sulphur  diox- 
ide is  oxidized  to  a trioxide  and  reaches  the  soil  as  free  sulphuric 
acid  or  'sulphate.  The  amount  of  sulphur  trioxide  brought  to 
an  acre  surface  yearly  has  been  determined  at  Rothamsted,  Eng- 
land. WaringtoiW  gives  the  amount  at  about  IT  pounds  and 
adds  further  that  sulphates  in  rains  “will,  to  a considerable  ex- 
tent, meet  the  demands  of  most  cultivated  crops.” 

Later  determinations  made  at  Rothamsted  and  furnished  us 
by  Director  Hall,  show  an  average  annual  precipitation  per 
acre  of  about  1 Sy2  pounds  of  sulphur  trioxide. 

In  England  large  amounts  of  soft  coal  are  burned.  It  was 
believed  not  improbable  that  the  amount  brought  to  a surface 
acre  annually  by  the  rain  in  the  open  country  of  our  northern 
states,  especially  where  soft  coal  is  used  to  but  a limited  extent, 
would  not  be  as  large  as  the  amount  found  in  England.  For 
the  purpose  of  collecting  data  on  this  point  a rain-gauge  with  a 
collecting  surface  equal  to  one  square  foot  was  set  up  at  the  Uni- 
versity Hill  Farm  located  three  miles  west  of  the  city  of  Wadi- 
son,  Wisconsin.  It  was  placed  in  an  open  field  and  at  least  a 
mile  from  any  chimney  using  considerable  quantities  of  coal. 
The  nearest  source  of  burning  coal  was  that  used  in  running  a 
stone  crusher  about  one  mile  east  of  the  location  chosen  for  the 
gauge.  As  the  prevailing  winds  in  this  part  of  the  country  are 
from  the  west  or  south,  the  direct  influence  of  this  chimney  as 
a source  of  sulphur  was  doubtless  minimized.  The  rain  water 
was  collected  in  a copper  bottle,  containing  a trace  of  sodium  bi- 
carbonate in  order  to  fix  any  sulphurous  acid.  A fine  wire 


ic  Chemistry  of  the  Farm,  9th  Ed.,  p.  19. 


Sulphur  Requirements  of  Farm  Crops 


15 


screen  was  placed  above  the  funnel  of  the  gauge  for  the  purpose 
of  excluding  any  material  accidentally  blown  or  falling  into  the 
gauge. 

The  samples  of  water  collected  were  analyzed  for  total  sul- 
phur trioxide  at  the  end  of  each  month.  For  this  determination 
the  water  was  evaporated  to  dryness  in  a platinum  dish,  treated 
with  bromine  and  nitric  acid,  again  evaporated  to  dryness,  the 
nitric  acid  displaced  by  evaporating  several  times  with  hydro- 
chloric acid  and  the*  sulphates  finally  precipitated  with  barium 
chloride.  The  results  are  given  in  Table  V and  reported  as 
pounds  per  acre  of  sulphur  trioxide ; the  monthly  rain  fall  at 
the  time  the  analyses  were  made  is  also  recorded. 


Table  V.—  Amount  in  Pounds  of  Sulphur  Trioxide  Broug-ht  to 
Surface  Acre  Monthly. 


June.  1910 

July.  1910  

A u trust.  1910 

September.  1910 
October.  1910.... 

Total 


Month. 


SOs 

Pounds. 

Rain  fall. 
Inches. 

2.3G 

1.31 

0.60 

0.81 

4.47 

6.56 

2.66 

1.83 

0.61 

0.63 

10.70 

11.14 

Not  enough  data  are  at  hand  to  warrant  establishing  an  aver- 
age annual  figure,  but  until  such  data  are  accumulated  the  ten- 
tative statement  can  be  made  that  the  amount  of  sulphur  tri- 
oxide brought  yearly  by  rain  to  an  acre  surface  of  land  at  this 
location  will  probably  not  be  less  than  that  found  at  Rotham- 
sted,  England,  which  is  17  to  18  pounds.  The  total  amount 
brought  down  in  the  five  months  recorded  was  10.70  pounds 
with  a rain  fall  of  11.11  inches.  Some  variations  in  the  sul- 
phur content  of  the  atmosphere  may  be  expected  from  season 
to  season,  as  well  as  variations  incident  to  frequency  of  rains; 
for  this  reason  a yearly  figure  must  be  based  on  actual  deter- 
minations over  that  period  rather  than  on  calculations  of  pre- 
cipitation per  inch  of  rain  fall  based  on  determinations  over 
only  short  periods  of  time. 


.16 


Wisconsin  Experiment  Station 


Losses  of  Sulphur  Trioxide  from  Soils  by  Drainage 

« 

While  the  atmosphere  can  serve  as  a considerable  source  of 
sulphur  trioxide  to  the  soil,  nevertheless  as  a compensating  fac- 
tor, it  is  probably  more  than  offset  by  the  losses  that  soils  sustain 
by  drainage.  It  is  well  known  that  most  river  and  lake  waters, 
which  in  part  represent  the  drainage  waters  from  our  soils,  con- 
tain considerable  quantities  of  sulphates.  These  have  been  dis- 
solved out  of  the  soil.  The  Kothamsted  Experiment  Station 
gives  some  definite  data  on  the  losses  of  sulphur  trioxide  in  the 
drainage  water  from  soils.  The  drainage  waters  from  the  wheat 
plots  of  Broadbalk  field  have  been  collected  from  time  to  time 
and  completely  analyzed  by  Yoelcker  and  Frankland.  From  the 
unmanured  plots  they  report  the  quantity  of  sulphur  trioxide 
in  the  drainage  water  at  24.7  parts  per  million,  while  the  quan- 
tity varied  from  41.0  to  106.1  parts  per  million  in  the  waters 
from  the  plots  receiving  various  fertilizer  treatments. 

Assuming,  as  Hall  does17  in  his  discussion  of  losses  by  drain- 
age from  these  plots,  a mean  annual  drainage  equal  to  10  inches 
of  water,  the  unmanured  plots  would  lose  approximately  50 
pounds  of  sulphur  trioxide  annually  per  acre,  while  those  re- 
ceiving fertilizers  containing  variable  amounts  of  sulphates 
would  lose  from  85  to  220  pounds  annually  per  acre.  It  will 
be  seen  from  the  above  data  that  the  loss  of  sulphur-  trioxide  by 
drainage  is  considerable  and  in  the  case  of  the  unmanured  plots 
it  is  nearly  treble  the  amount  brought  by  rain  to  an  acre  sur- 
face from  the  atmosphere.  While  these  figures  may  not  be  ap- 
plicable to  all  climates  and  all  soils,  nevertheless  it  would  be 
conservative  to  state  that  the  loss  by  drainage  at  least  equals 
and  probably  exceeds  the  amount  brought  to  the  soil  from  the 
atmosphere  in  the  humid  regions  of  America. 

General  Discussion. 

The  general  question  raised  by  the  data  presented  above  is 
one  of  great  importance.  The  fact  that  common  crops  remove 
from  the  soil  considerable  quantities  of  the  element  sulphur, 
while  the  compensating  factor  of  supply  from  the  atmosphere 
is  very  probably  offset  by  the  losses  which  the  land  sustains  by 


.17  The  Soil,  p.  200. 


Sulphur  Requirements  of  Farm  Crops 


17 


drainage,  makes  it  apparent  that  for  the  maintenance  of  a per- 
manent supply  of  sulphur  in  the  soil,  this  element  must  be 
added  systematically  either  as  a constituent  of  commercial 
fertilizers,  or  with  the  farm  manure. 

The  supply  of  sulphur  in  soils  is  low,  the  surface  eight  inches 
of  a normal  soil  containing  sulphur  trioxide  sufficient  for  about 
100  crops  of  barley,  and  this  would  suppose  that  the  crop  could 
produce  normal  yields  with  a steadily  decreasing  supply  of 
sulphur.  The  upward  movement  of  water  within  the  soil  occa- 
sioned by  surface  evaporation,  would  possibly  aid  in  bringing 
sulphates  from  the  lower  soil  areas,  but  this  could  not  continue 
indefinitely.  The  sulphur  trioxide  content  of  sub-soils  accord- 
ing to  the  analyses  made  by  Hilgard  is  no  greater  than  that  of 
the  surface  soil.  Many  factors  are  operative  in  the  maximum 
production  of  crops ; ample  supplies  of  essential  elements,  proper 
biological  agents,  absence  of  toxic  substances,  and  proper  physi- 
cal environment  are  all  important  soil  factors.  So  many  fac- 
tors are  operative  that  an  optimum  condition  is  probably  seldom 
obtained.  For  this  reason  the  disturbance  of  the  equilibrium 
in  the  soil  by  the  addition  of  a single  agent  is  often  followed  by 
better  crop  production.  Whether  it  affects  the  biological  agents, 
improves  the  sanitary  conditions,  alters  the  physical  status  of 
• the  soil,  or  acts  by  furnishing  an  ample  supply  of  the  elements 
essential  for  plant  growth  is  always  difficult  to  answer,  but  that 
there  must  be  maintained  an  ample  supply  of  the  essential  ele- 
ments. is  one  of  the  first  principles  of  plant  production. 

Most  normal  soils  contain  an  abundance  of  the  essential  ele- 
ments, potassium,  iron,  magnesium  and  calcium.  Nitrogen, 
phosphorus  and  sulphur  alone  are  percentagely  low  and  in  a 
class  by  themselves.  The  former  two  are  today  recognized  as 
valuable  essentials  of  commercial  fertilizers  and  manures, 
and  field  experiments  in  many  instances  have  shown  the 
utility  of  the  application  of  potash  salts  in  available  form. 
When  calcium  is  added  it  is  usually  as  a carbonate  and  more 
for  the  purpose  of  maintaining  a neutral  or  slightly  alkaline 
reaction  in  the  soil  solution  than  as  a source  of  needed  calcium 
for  plant  growth. 

The  use  of  sulphates  in  fertilizers  has  been  unconsciously 
practiced  for  many  years.  The  acid  phosphate  of  the  fertilizer 
industry  is  a product  containing  a large  proportion  of  calcium 
sulphate.  Whether  the  beneficial  results  accruing  from  its  ap- 
plication are  to  be  attributed  alone  to  the  phosphorus  it  supplies 


18 


Wisconsin  Experiment  Station 


or  whether  they  are  twofold  and  due  to  both  the  phosphorus  and 
sulphur  contained  in  this  material  are  questions  raised  by  this 
investigation.  It  is  not  impossible  that  the  superior  results 
sometimes  obtained  in  field  practice  with  acid  phosphates  over 
other  phosphates,  such  as  Thomas  Slag,  or  ground  rock  phosph- 
ates, are  not  due  entirely  to  a difference  in  solubilities,  but  to  the 
additional  sulphur  supplied  by  the  former  material.  The  supe- 
rior results  so  often  noted  from  the  use  of  potash  salts  when  fur- 
nished as  sulphates  rather  than  chlorides,  may  rest  in  part  upon 
the  sulphur  content  of  the  material  added. 

It  was  once  common  practice  to  use  gypsum  as  a fertilizer. 
The  beneficial  results  often  resulting  from  the  use  of  this  ma- 
terial, have  been  explained  on  the  basis  of  its  action  as  a stimu- 
lant. Boussingault  showed  that  its  application  increased  the 
amount  of  potassium  taken  up  by  the  plant.  This  was  accom- 
plished through  a double  decomposition  of  potassium  silicates 
with  liberation  of  potassium  sulphate.  Consequently  its  action 
was  said  to  be  indirect  and  it  was  classed  as  a stimulant  for  the 
reason  that  while  it  often  produced  increased  plant  growth,  it 
furnished  no  necessary  plant  elements.  While  the  above  reaction 
may  be  entirely  true,  the  explanation  appears  to  be  only  partial. 
If  the  idea  here  presented,  that  sulphur  may  become  a limiting 
element  in  crop  production,  is  true,  then  the  beneficial  results  • 
accruing  from  the  use  of  gypsum  may  result  in  part  from  its 
sulphur  content. 

In  the  25  years  of  carefully  recorded  plot  fertilization  experi- 
ments by  the  Pennsylvania  Experiment  Station,18  the  evidence 
for  the  necessity  of  occasional  sulphur  applications  is  negative. 
While  these  experiments  were  not  planned  to  answer  the  ques- 
tion of  sulphur  requirements,  nevertheless  they  are  the  only 
experiments  available,  so  far  as  the  authors  are  aware,  which 
shed  any  light  at  all  on  the  question  involved.  On  plots  9 and 
17  the  treatment  consisted  of  dried  blood,  muriate  of  potash 
and  dissolved  bone  black,  which  probably  contained  calcium 
sulphate  sufficient  to  furnish  from  30  to  40  pounds  of  sulphur 
trioxide  per  acre.  On  plots  12  and  35  the  treatment  was  dried 
blood,  muriate  of  potash  and  ground  bone.  Identical  quantities 
of  phosphorus  pentoxide  and  potassium  oxide  were  added  in  the 


38  Rep.  Pa.  Expt.  Sta.,  1907-1908,  p.  80. 


Sulphur  Requirements  of  Farm  Crops 


19 


two  treatments,  with  24  pounds  of  nitrogen  in  the  first  mixture 
and  30  pounds  in  the  latter. 

Unless  the  muriate  of  potash  and  dried  blood  used  contained 
sulphates  sufficient19  for  all  crop  needs,  then  the  only  difference 
in  the  kinds  of  essential  elements  added  was  in  the  greater 
amount  of  sulphur  contained  in  the  first  mixture ; yet  the  total 
annual  yield  in  crops  over  a period  of  25  years  was  slightly  in 
favor  cf  the  second  treatment.  Nevertheless,  while  an  annual 
removal  of  15  pounds  of  sulphur  trioxide  per  acre  by  the  crops 
grown  in  the  above  experiment,  may  not  as  a single  factor  be 
sufficient  to  reduce  the  productivity  of  the  soil  receiving  suffi- 
cient nitrogen,  phosphorus  and  potassium  in  25  years,  it  appears 
probable  that  this  could  not  continue  indefinitely.  Our  own 
data  on  the  partial  depletion  of  sulphur  in  soils  unmanured  but 
cropped  for  50  to  60  years  is  evidence  in  support  of  this  view. 

Unfortunately  none  of  the  experimental  plots  at  Rothamsted, 
England,  have  been  deliberately  planned  with  respect  to  the 
effect  of  fertilizers  with  and  without  applications  of  sulphur 
on  long  continued  cropping.  We  are,  however,  through  the 
courtesy  of  Director  Hall,  in  possession  of  materials  from  that 
station  the  analysis  of  which  may  throw  additional  light  on 
the  question  involved.  Such  data  will  be  reported  later. 

Careful  experimentation  and  practical  agriculture  must  de- 
cide in  what  form  sulphur,  when  needed,  should  be  added  to  the 
soil.  Economy  and  safety  are  the  factors  involved.  The  prin- 
cipal sulphates  normal  to  the  soil  are  those  of  calcium,  mag- 
nesium and  potassium.  No  harm  can  result  from  a judicious 
use  of  any  of  these.  Sulphur  can  be  added  to  the  soil  either 
as  land  plaster;  with  acid-phosphate,  in  which  it  exists  as  cal- 
cium sulphate ; or  as  a sulphate  of  potassium  or  ammonium.  All 
these  materials  are  now  offered  by  the  trade.  A ton  of  land 
plaster  contains  about  900  pounds  of  sulphur  trioxide  and  a ton 
of  acid-phosphate  will  carry  from  200  to  300  pounds  of  sulphur 
trioxide,  in  addition  to  the  phosphorus  pentoxide  it  contains.  A 
ton  of  high  grade  sulphate  of  potassium  will  contain  about  900 
pounds  of  sulphur  tri oxide  besides  about  1,000  pounds  of  potas- 
sium oxide.  A ton  of  ammonium  sulphate  will  contain  about 


is  From  some  of  our  own  analyses  of  dried  blood  and  muriate  of  pot- 
ash the  amounts  of  these  materials  used  bi-annually  in  the  Pennsyl- 
vania Experiments  could  have  furnished  about  14  pounds  of  sulphur’ 
trioxide  per  acre. 


20 


Wisconsin  Experiment  Station 


1,000  pounds  of  sulphur  trioxide  besides  its  large  amount  of 
nitrogen. 

Of  course,  under  systems  of  stock  farming  where  the  crops 
and  purchased  feeds  are  fed  and  the  manure  saved,  the  sulphur 
will  find  its  way  back  to  the  land,  but  whether  even  then  the- 
losses  by  drainage  and  in  the-  practical  handling  of  the  manure 
must  not  be  met  by  additional  applications  of  sulphates,  are 
questions  still  to  be  determined.  In  systems  of  grain  farming 
it  appears,  from  the  data  here  presented,  that  some  form  of 
sulphate  must  be  used  systematically  in  the  fertilizer  treat- 
ment of  the  soil  for  the  purpose  of  maintaining  therein  a per- 
manent supply  of  the  element  sulphur. 

Conclusions. 

1.  The  sulphur  content  of  a number  of  our  common  farm 
products  has  been  determined  and  in  agreement  with  other  in- 
vestigations the  quantity  is  much  larger  than  found  by  Wolff 
in  the  ash  from  such  products. 

2.  The  amount  of  sulphur  trioxide  removed  by  crops  is  con- 
siderable, being  equal  in  the  case  of  average  crops  of  cereal 
grains  and  straws  to  about  two-thirds  of  the  phosphorus  pent- 
oxide  removed  by  these  crops;  the  grasses  of  mixed  meadow  hay 
remove  quite  as  much  sulphur  as  phosphorus,  while  the  legume 
hays  may  approach,  and  in  the  case  of  alfalfa,  even  exceed  in  this 
respect.  Members  of  the  Cruciferae,  as  the  cabbage  and  turnip, 
are  heavy  sulphur-using  crops  and  may  remove  two  to  three 
times  as  much  sulphur  trioxide  as  phosphorus  pentoxide.  An 
average  acre  crop  of  cabbage  will  remove  about  100  pounds  of 
sulphur  trioxide. 

3.  Normal  soils  are  relatively  poor  in  total  sulphur  trioxide; 
a limited  number  of  analyses  showed  a percentage  content  of 
from  0.033  to  0.140;  most,  of  them  contained  less  than  0.10  per 
cent.  An  acre  foot  will  contain  from  1,000  to  3,000  pounds  of 
total  sulphur  trioxide.  About  the  same  quantity  of  phosphorus 
pentoxide  will  be  found  in  an  acre  foot  of  normal  soil.  These 
results  for  sulphur  trioxide  are  based  on  analyses  made  by  the 
method  of  fusion  with  sodium  peroxide.  Determinations  by 
extracting  with  hydrochloric  acid  or  with  nitric  acid  and  bro- 
mine will  not  give  the  total  sulphur  content  of  soils. 

4.  Soils  cropped  for  50  to  60  years  and  either  unmanured  or 


Sulphur  Requirements  of  Farm  Crops 


21 


receiving  but  slight  applications  during  that  period  have  lost 
on  the  average  40  per  cent  of  the  sulphur  trioxide  originally 
present  as  determined  by  comparison  with  virgin  soils. 

5.  Where  farm  manure  has  been  applied  in  regular  and  fairly 
liberal  quantities  the  sulphur  content  of  the  soil  has  been  main- 
tained and  even  increased. 

6.  The  total  sulphur  trioxide  precipitated  at  Madison,  Wis., 
with  the  rain  amounted  in  the  five  months  of  June  to  October, 
1910,  inclusive,  to  11.7  pounds  per  acre.  The  annual  amount 
may  tentatively  be  placed  at  from  15  to  20  pounds. 

7.  The  losses  of  sulphur  trioxide  by  drainage,  based  on  the 
analysis  of  the  drainage  waters  at  Rothamsted,  England,  and  on 
a yearly  drainage  of  10  inches,  would  amount  to  about  50 
pounds  per  acre'  yearly. 

8.  Even  with  much  less  loss  by  drainage  it  does  not  appear 
that  the  atmosphere  can  serve  as  a complete  compensating  fac- 
tor for  losses  of  sulphur  trioxide  which  soils  sustain  through 
both  cropping  and  drainage.  The  partial  depletion  of  the  sul- 
phur of  the  soil  by  continued  cropping  without  adequate  fertil- 
ization is  evidence  in  support  of  this  view. 

9.  From  the  data  here  presented  it  appears  that  for  perma- 
nent and  increased  production  of  farm  crops  such  systems  of  fer- 
tilization must  be  inaugurated  as  will  supply  to  the  soil  from 
time  to  time,  in  addition  to  the  elements  nowT  recognized  as 
generally  necessary, — namely,  nitrogen  and  phosphorus, — a 
sufficient  quantity  of  sulphur  to  meet  the  losses  sustained  by 
cropping  and  drainage. 

10.  Such  sources  of  sulphur  are  farm  manures ; the  trade  fer- 
tilizers, such  as  super-phosphate,  ammonium  sulphate  and  sul- 
phate of  potassium;  and  the  so-called  soil  stimulant,  gypsum  or 
calcium  sulphate. 

In  a problem  of  such  large  significance  the  authors  realize  the 
desirability  of  extreme  caution  and  conservatism  in  presenting 
the  views  outlined.  No  attention,  so  far  as  we  are  aware,  has 
been  directed  to  this  problem  in  America.  It  is  hoped  that  the 
thesis  here  presented  may  be  made  the  subject  for  further  re- 
search by  chemists  and  agronomists  and  the  relative  importance 
and  necessity  for  sulphur  in  systems  of  fertilization  finally 
established. 


f jfT 

Experiments  on  Spore  Germination  and 
Infection  in  Certain  Species 
of  Oomycetes1 


I.  E.  MELHUS 

INTRODUCTION 

Some  two  years  ago  special  work  was  undertaken  upon  Cysto- 
pus  and  other  Oomycetes  aiming  to  learn  more  as  to  the  methods 
of  spore  germination,  zoospore  formation,  methods  of  infection, 
and  as  to  the  possible  existence  of  so-called  “physiological  spe- 
cies” in  the  genus  Cystopus.  The  parasite  Cyst  opus  candidus 
was  common  on  the  garden  crop  of  radish,  Eaphanus  sativus, 
and  attempts  were  made  at  the  outset  to  produce  infection  by 
transferring  spores  from  this  to  radishes  growing  in  the  green- 
house. Although  repeated  trials  were  made  in  connection  with 
these  early  studies,  only  meager  and  irregular  infections  resulted. 
This  suggested  that  some  variable  factors  of  unknown  nature 
were  present  in  the  greenhouse  trials.  Difficulty  was  also  en- 
countered at  the  outset  in  securing  uniform  results  in  spore 
germination  by  the  methods  described  by  earlier  workers.  Thus 
it  soon  became  evident  that  some  more  specialized  methods  were 
necessary  in  order  to  secure  the  germination  of  the  conidia  and 
host  infection  in  abundance  and  with  a satisfactory  degree  of 
certainty. 

We  were  thus  led  to  attempt  to  determine  the  relations  of 
* various  conditions  to  spore  germination  and  infection  with  this 
fungus,  not  only  host  relations,  but  also  relations  of  the  age 

1 The  author  wishes  to  express  to  Dr.  R.  A.  Harper  and  Dr.  L.  R. 
Jones  his  sincere  appreciation  for  the  kind  criticism  and  keen  interest 
shown  during  the  progress  of  tbi$  work  and  preparation  of  the  mamj. 
script, 


26 


Wisconsin  Experiment  Station. 


and  maturity  of  the  spores,  moisture,  food,  chemical  stimuli,  light 
and  temperature.  Other  species  were  subsequently  tested.  The 
results  secured  are  of  such  definiteness  and  breadth  of  applica- 
bility as  to  justify  their  publication,  although  much  remains  to  be 
done  upon  the  problems  as  originally  defined.  Before  discussing 
my  own  work  and  conclusions,  a brief  review  will  be  made  of  the 
results  of  previous  studies  upon  these  and  closely  related  mat- 
ters. 

Review  of  Earlier  Work 

The  germination  of  the  asexual  spores  of  Cystopus  was  first 
observed  over  a century  ago  by  Prevost  (1807 :29).  He  studied 
the  species  commonly  parasitic  on  crucifiers  and  purslane 
respectively,  then  known  as  TJredo  Candida  and  TJredo  portulacae. 
His  description  is  clear  and  interesting.  He  states  that  the 
spores  germinated  one  or  two  hours  after  immersion,  some- 
times within  40  or  45  minutes,  owing  in  all  probability  to  differ- 
ences in  temperature,  which,  during  the  observation,  fluctuated 
between  12°  and  16°  R.  (equivalent  to  15°  and  20d  C.).  In  the 
process  of  germination  the  spores  absorb  water  and  become  bottle 
shaped;  soon  a globule  (zoospore)  is  seen  on  the  outside  and  this 
is  immediately  followed  by  several  others,  sometimes  as  many  as 
six  more.  He  states  that  these  globules  instantly  reunite  into  a 
mass  which  moves  as  a unit  by  rolling  about  in  the  water.  The 
globules,  as  a rule,  separate  from  one  another  in  a very  short 
time ; sometimes,  however,  two  or  even  three  globules  may  remain 
attached  together,  either  immediately  touching  or  as  if  joined  by 
a string.  Those  globules  which  separate  from  one  another,  and 
they  are  by  far  the  greater  number,  are  sometimes  a little  angular 
and  possibly  a little  hollowed  or  pushed  in  at  one  side.  They 
swim  about  in  the  same  way  as  when  united  in  mass.  Soon  the 
movement  of  the  globules  ceases  and  they  become  fixed  at  the 
surface  of  the  water  or  at  the  edge  of  the  drop.  He  observed 
that  the  swarmspores  developed  germ  tubes,  regarding  which 
however  he  gives  little  detail.  Prevost  likewise  studied  the 
Cystopus  on  salsify  and  Amaranthus  and  found  the  last  two# 
forms  much  more  difficult  to  germinate. 

Tulasne  (1854:77)  states  that  he  germinated  the  spores  of 
tiredo  portulacae  and  TJredo  Candida  but  was  unable  to  get  them 
to  form  swarmspores  in  the  manner  described  by  Prevost,  find- 
ing ofily  the  germination  by  a tube. 


Experiments  on  Spore  Germination. 


27 


Hoffman  (1859 : 210)  was  also  unable  to  confirm  Prevost  as  to 
the  formation  of  swarm  spores  in  Cystopus.  He  describes  the 
germination  of  the  spores  by  tubes  in  the  same  manner  as 
described  by  Tulasne. 

DeBary  (I860:  236)  studied  the  method  of  germination  in 
Cystopus  cubicus  and  Cystopus  candidus  and  found  the  germina- 
tion was  by  zoospores,  as  described  by  Prevost,  but  that  germina- 
tion might  take  place  at  any  temperature  between  5°  and 
25°,  C.  He  emphasized  for  the  first  time  that  the  spores  are 
really  sporangia  producing  from  five  to  eight  zoospores  in  Cysto- 
pus candidus  and  from  eight  to  twelve  in  Cystopus  cubicus. 
DeBary  describes  the  changes  in  the  sporangium  very  clearly. 
The  spores  absorb  water  and  swell  when  sown  in  a drop  of  water. 
On  one  side  an  obtuse  papilla  is  developed  and  vacuoles  form  in 
the  granular  protoplasm  which  disappear  later.  At  this  stage  of 
development,  fine  lines  of  demarcation  divide  the  protoplasm 
into  five  to  eight  polyhedral  portions  leaving  at  the  center  a small 
feebly  colored  vacuole.  When  the  division  of  the  content  of  the 
sporangium  is  complete,  the  papilla  swells,  opens  and  the  zoo- 
spores are  pushed  to  the  outside  one  by  one,  showing  no  sign  of 
movement.  Once  outside  the  sporangium  they  become  lenticular 
in  form  and  group  themselves  before  the  opening  of  the  sporan- 
gium in  a spherical  mass.  Yery  soon  the  swarm  spores  begin  to 
move,  vibratile  cilia  appear  and  the  globular  mass  is  set  oscillat- 
ing. The  zoospores  ultimately  become  free  and  swim  away  singly. 
The  motile  spores  are'  plano-convex  or  slightly  concavo-convex 
having  a small  disk-like  vacuole  on  one  side.  Attached  near  the 
vacuole  are  two  cilia,  a short  one  in  front  and  a long  one  behind, 
both  on  the  same  side.  In  from  one  and  a half  to  three  hours 
after  being  placed  in  water  the  escape  of  the  zoospores  begins. 
They  will  develop  either  from  sporangia  freshly  formed  or  from 
those  which  have  been  kept  as-  long  as  six  weeks. 

DeBary  (1863:14)  found  the  conidia  of  Cystopus  germ- 
inating on  the  leaves  of  the  host  plants.  Zoospores  were  found 
in  the  drops  of  water  on  the  leaves.  Infection  experiments  with 
Cystopus  candidus  were  made  on  various  hosts.  In  the  case  of 
Lepidium  sativum  the  zoospores  readily  entered  the  stomata  of 
both  leaves  and  cotyledons  but  produced  infection  only  in  the 
latter.  Various  species  of  Brassica  showed  the  same  tendency, 
though  not  to  so  marked  an  extent. 


28 


Wisconsin  Experiment  Station. 


Farlow  (1875:319)  studied  the  germination  of  the  conidia  of 
Phytophthora  infestans  and  observed  that  sometimes  the  con- 
tents of  the  spore  discharged  in  one  mass,  and  from  this  mass 
zoospores  are  produced  as  before.  He  believes  that  the  produc- 
tion of  zoospores  is  favored  by  darkness,  whereas  germination  by 
a germ  tube  takes  place  more  frequently  in  the  light.  He  states, 
however,  that  he  has  repeatedly  sown  spores  in  watch  glasses  and 
both  methods  of  germination  resulted.  The  germinating  power 
of  the  spores  was  retained  for  several  weeks,  but  they  did  not 
germinate  after  a winter’s  exposure. 

Farlow  (1876  :419)  also  describes  the  germination  of  the  conidia 
of  Plasmopara  viticola.  During  the  month  of  October  when  the 
disease  is  most  prevalent,  he  found  that  in  one  and  one-fourth 
hours  zoospores  were  formed  and  began  to  make  their  way  to  the 
outside  of  the  sporangium.  The  conidia  might  also  germinate 
directly,  i.  e.,  by  germ  tubes.  Darkness  was  more  favorable  for 
the  germination  of  the  conidia,  whether  directly  or  indirectly. 
He  found  the  zoospores  to  swim  about  for  fifteen  to  twenty  min- 
utes, after  which  the  motion  gradually  became  slower  until  they 
finally  came  to  rest.  In  another  quarter  of  an  hour  an  outgrowth 
appeared  on  one  side  which  rapidly  developed. 

Scribner  (1886:10)  states  that  temperature  exercises  a consid- 
erable influence  over  the  germination  of  Plasmopara  viticola  from 
the  grape.  The  most  favorable  temperature  lay  between  25°  and 
35°  C.  At  a lower  temperature,  germination  took  place  more 
slowly,  but  the  temperature  could  be  reduced  to  zero  without 
destroying  the  vitality  of  the  conidia.  Under  exceptional  cir- 
cumstances, Scribner  states,  another  form  of  germination  might 
occur  in  which  a conidium  may  push  out  a tube.  This  method, 
he  reports  as  undoubtedly  rare. 

The  question  of  spore  germination  and  physiological  species  in 
the  genus  Cystopus  has  quite  recently  been  studied  in  consid- 
erable detail  and  with  very  interesting  results  by  Eberhardt 
(1904:622).  He  began  his  work  in  June  1902  and  continued 
it  until  the  fall  of  1903.  Cystopus  candidus  on  various  crucifer- 
ous plants  were  used  except  in  one  experiment  where  he  used 
Cystopus  cubicus  on  Tragopogon  pratensis. 

Eberhardt  (1903:655)  found,  as  we  have,  considerable  diffi- 
culty in  germinating  the  conidia  of  Cystopus.  He  tried  the  dif- 
ferent methods  used  by  DeBary  and  Zalewski,  but  obtained  only 
a low  germination.  To  solve  the  question  of  spore  germination, 


Experiments  on  Spore  Germination. 


29 


Eberhardt  turned  his  attention  to  methods  of  properly  maturing 
me  comma,  inuring  ail  die  montii  of  iviay  ana  xhe  beginning 
of  June  Cyst  opus  cantiidus  was  available  on  Capsella,  twelve 
experiments  were  inaae  at  as  many  different  times  of  which 
tlie  following  may  be  taken  as  typical.  (Jonidial  pustules  not 
opened,  were  collected  from  time  to  time.  The  contents  of 
die  pustules  Were  placed  in  a vial  containing  a small  amount 
of  rain  water.  This  was  called  vial  No.  1.  In  vial  No.  2, 
containing  ram  water  was  placed  the  spores  shaken  from  open 
pustules,  in  still  another  test  a shoot  was  taken  which  bore 
many  unopened  pustules.  It  was  wrapped  in  a moist  cloth  to 
be  kept  for  furtner  observations. 

In  vial  No.  1 the  conidia  remained  in  chains,  the  protoplasm 
became  granular  and  later  bacteria  developed  decomposing  the 
conidia.  Vial  No.  1 may  show  a small  number  of  conidia  ger- 
minated, but  the  larger  part  disorganized.  In  vial  No.  2, 
containing  spores  shaken  from  open  pustules  one  third  of  the 
conidia  formed  zoospores  while  the  remainder  decomposed. 
ATien  taking  spores  from  pustules  just  opened,  more  than  one 
half  germinated.  It  is  permissible  to  think  that  the  former 
were  relatively  old  and  that  they  had  lost  their  capacity  of 
germination  whereas  the  later  conidia  were  taken  from  pustules 
just  opened  and  gave  a germination  of  more  than  one  half 
of  the  spores.  The  third  test,  in  which  the  shoot  containing 
pustules  was  wrapped  in  a moist  cloth,  gives  further  evidence 
in  support  of  the  above  interpretation.  After  the  pustules  had 
been  kept  moist  for  one  day  a microscopic  examination  of  three 
of  the  pustules  showed  the  following  conditions  on  being  placed 
in  a drop  of  water.  The  young  pustule  was  found  to  contain 
the  zoosporangia  in  chains  which  gave  no  germination.  The 
second  showed  a small  fraction  of  the  spores  empty  or  bottle- 
necked, ready  to  germinate.  The  other  conidia  were  decom- 
posing. The  third  presented  all  the  conidia  disassociated  and 
about  one  third  produced  zoospores.  The  shoot  was  kept  fur- 
ther until  almost  all  of  the  pustules  had  opened  liberating  the 
spores  that  germinated  in  fairly  large  numbers.  This  same 
shoot  was  kept  two  weeks  longer  when  it  had  wilted  and  dried 
up  in  places.  The  conidia  that  fell  from  it  gave  no  germination. 

These  experiments  show  us  that  the  zoosporangia  are  the 
organs  of  immediate  infection,  requiring  for  germination  to  be 
collected  at  the  time  when  the  pustules  open.  Infection  occurs 


30  Wisconsin  Experiment  Station. 

when  these  spores  fall  upon  the  young  host  plants  humid  from 
rain  or  dew.  In  the  numerous  cultures  made  for  infection 
purposes  none  were  kept  after  the  third  day  if  germination 
had  not  already  occured.  It  is  well  here  to  notice  the  variation 
in  time  required,  for  germination  of  conidia.  A few  notes  from 
Eberhardt’s  experiments  will  suffice. 

Eberhardt  used  “room  temperature”  in  every  case  except 
in  one.. test  made  on  the  twenty-ninth  of  March.  The  exact 
temperatures  are  given  in  only  two  cases,  however.  In  the 
first  it  varied  from  11°  to  17°  C.  In  the  experiment  referred 
to,  on  March  29,  the  temperature  varied  from  2°  to  8°  C.  The 
latter  condition  was  obtained  by  placing  the  culture  on  a win- 
dow sill  where  the  influence  of  the  out-door  temperature  had 
some  effect.  His  germination  experiments  that  are  described 
were  carried  on  from  March  28  to  August  9,  1902,  on  five 
different  dates,  all  resulting  in  germination  in  from  three  to 
forty  hours. 

There  was  also  another  difficulty  greater  than  that  of  the 
selection  of  the  conidia,  it  was  the  choice  of  the  proper  time 
to  inoculate  the  host  plant.  The  task  of  growing  and  caring 
for  the  young  plants,  the  delicacy  of  the  material  of  infection 
and  the  considerable  space  required  for  growing  the  crucifers 
has  made  it  impossible  to  often  repeat  the  same  infections. 

In  view  of  the  fact  that  the  following  statements  of  the 
author  are  not  clear  we  quote  directly.  “Un  facteur  important 
etait  la  coincidence  qui  doit  tou jours  exister  entre  la  recolte  du 
parasite  et  l’etat  de  germination  propice  de  la  plante  a infecter. 
C’est  pour  ces  raisons  que  nous  ne  pouvons  poser  en  ce  moment 
aucune  regie  basee  sur  l’experience,  relative  a 1 ’optimum  de 
receptivite  du  parasite  par  le  vegetal  nourricier.  Mais  ce  que 
nous  pouvous  certainement  avancer,  c’est  qu’il  ne  suffit  pas  dans 
toutes  les  Crucifers  d ’avoir  des  cotyledons  bien  etales.  La  ques- 
tion de  1 ’optimum  de  receptivite  demande  plusieurs  annees  de 
recherches.  Comme  nos  infections  tendaient  plutot  a prouver 
1 ’unite  d’espece,  ce  n’est  que  tres  tard  que  nous  nous  sommes 
aperu,  apres  des  insucces  nonib reux,  combien  l’etat  du  jeune 
plant  influe  sur  la  reussite  de  l’experience.  Ainsi  nous  avons 
vu  que  certains  cotyledons,  sortant  de  la  game  refusent  1 ’entree 
du  parasite,  tandis  que  lorsqu  ’ils  sont  bien  etales,  ils  sont  sus- 
ceptibles  d ’infections.  II  nous  a semble  que  plusieurs  de  nos 
especes  qui  avaient  ete  infectees  au  moment  ou  les  cotyledons 


Experiments  on  Spore  Germination. 


31 


etaient  fanes  penvent  recevoir  Fendophyte  par  le  jeune  bourgeon 
foliaire  deroulant  ses  feuilles.  Mais  nous  ne  pouvons  encore  rien 
affirmer  a ce  propos.  Au  reste,  De  Bary  lui-meme  indique  que 
Heliophila  crithmifolia  est  apte  a etre  infectee  par  les  jeunes 
feuilles.  ’ ’ 

Comparatively  speaking,  but  very  little  work  has  been  done  on  * 
the  question  of  physiological  species  in  the  Oomycetes.  Eber- 
hardt  (1904:714)  has  investigated  this  problem  in  Cystopus  can- 
didus  occurring  on  various  crucifers.  Plants  were  inoculated  by 
germinating  the  conidia  and  placing  the  liquid  containing  the 
zoospores  on  the  lower  side  ’of  the  cotyledons  of  the  plants  to  be 
infected  or  simply  dipping  the  cotyledons  to  be  infected  in  the 
water  containing  the  zoospores.  The  seedlings  were  grown  in 
large  flower  pots  and  small  bunches  were  removed  as  needed  for 
infection.  His  infection  experiments  were  carried  on  both,  out  of 
doors  and  in  an  ordinary  laboratory.  The  conidia  of  five  different 
species  were  used  as  material  for  infecting  other  cruciferous 
plants. 

It  is  further  interesting  to  note  that  Eberhardt  made  inocula- 
tion experiments  with  the  oospores  of  Cystopus  candidus  from 
Lepidium  sativum.  In  order  to  infect  the  plants  of  Lepidium 
and  Capsella  with  the  oospores  from  Lepidium  sativum,  the  parts 
of  the  host  containing  the  oospores  were  placed  in  small  bags  and 
hung  out  of  doors  in  the  open  air  during  the  winter  months.  In 
March  and  April  the  oospore  material  was  distributed  over  the 
surface  of  pots  where  it  might  decay  and  liberate  the  oospores. 
Two  of  these  pots  were  then  seeded  with  Lepidium  sativum  and 
Capsella  Bursa-pastoris  and  the  young  seedlings  became  infected. 
It  should  be  noted  in  this  connection  that  Eberhardt  used  no  con- 
trols in  this  series  of  experiments. 

His  results  may  be  summarized  as  follows:  With  the 

conidia  of  Cystopus  candidus  from  Capsella  Bursa-pastoris  he 
infected  Capsella  Bursa-pastoris.  Lepidium  sativum , Iberis 
amara,  and  Arabis  alpina.  Conidia  from  Capsella  Heegeri 
infected  Capsella  Heegeri,  Capsella  Bursa-pastoris  and  Lepidum 
sativum.  Conidia  from  Lepidium  sativum  infected  Lepidium 
sativum  and  Capsella  Bursa-pastoris.  Spores  from  Brassica 
rapa  infected  Brassica  rapa,  Brassica  oleracea  (varieties: 
botrytis,  capitata,  and  congylodes),  Brassica  nigra,  Sinapis 
arvensis , and  Diplotaxis  tenuifolia.  The  widest  range  of  infec- 


32 


Wisconsin  Experiment  Station. 


tion  was  obtained  with  the  conidia  from  Arabis  alpina  which 
infected  Arabis  alpina , Arabis  hirsuta,  Arabis  II  alter  i,  Arabis 
turritis,  Lepidium  sativum,  Iberis  amara,  Cardamine  pra- 
tensis,  Cardamine  amara,  Capsella  Bursa-past ons,  and  Sene- 
biera  eoronopus.  With  the  oospores  from  Lepidium  sativum 
he  infected  Lepidium  sativum  and  Capsella  Bursa-past  oris.  It 
was  impossible  to  infect  any  of  the  Cruciferae  with  conidia  from 
Tragopogon  pratensis,  but  quite  easy  to  infect  Scorzonera  Jus 
panica.  From  these  data  Eberhardt  believes  that  there  are  no 
biological  species  in  the  species,  Cyst  opus  candidus.  It  should 
be  noted,  however,  that  the  above  conclusions  are  based 
upon  only  one  trial  in  some  cases  with  each  species  or 
variety  of  plant.  Eberhardt  states  in  this  connection  that  the 
laborious  task  of  growing,  inoculating  and  recording  results, 
together  with  the  fact  that  the  ground  used  for  growing  the 
plants  could  not  be  had  during  the  next  year,  made  it  impossible 
to  duplicate  any  of  the  series  of  experiments  performed. 

The  fact  that  Eberhardt ?s  work  was  incomplete  and  not  fully 
convincing,  and  that  it  is  especially  important  that  each  group 
of  parasitic  fungi  be  fully  understood  as  to  the  existence  of 
physiological  species,  led  me  to  study  this  problem. 


EXPERIMENTAL  STUDIES  IN  SPORE  GERMINATION 

Method 

As  has  been  previously  stated,  the  major  portion  of  the  experi- 
ments recorded  in  this  paper  were  carried  on  with  the  common 
white  rust,  Cyst  opus  candidus,  as  it  occurs  on  various  garden 
plants.  The  culture  work  was  all  carried  on  either  in  the  green- 
house or  in  an  ordinary  laboratory.  Both  distilled  and  tap  water 
were  used  to  germinate  spores.  The  tap  water  in  this  case  is  un- 
filtered water  drawn  directly  from  Lake  Mendota.  The  conidia 
were  gathered  from  infected  plants  growing  out  of  doors  until 
frost,  when  they  were  taken  from  infected  plants  in  the  green 
house  and  were  sown  the  same  day  as  gathered.  The  spores  were 
sown  in  a drop  of  water  placed  near  the  center  of  a clean  slide.  It 
was  always  difficult  to  make  the  conidia  sink,  but  by  stirring  the 
drop  with  a scalpel,  a large  per  cent  could  be  finally  made  to  settle 


Experiments  on  Spore  Germination. 


33 


to  the  bottom.  The  aim  was  always  to  add  only  a moderate  num- 
ber of  spores  to  each  drop,  since  too  many  spores  make  the  water 
opaque  and  difficult  to  examine.  Care  was  taken  to  obtain  fresh 
spores  in  every  case,  which  were  stirred  into  the  drop  of  tap 
water.  Sometimes  small  pieces  of  leaves  bearing  pustules  were 
dropped  into  water  on  a slide. 

The  first  experiments  were  made  at  room  temperature,  but  the 
irregularity  of  the  results  and  the  small  per  cent  of  the  total 
which  germinated  even  in  the  most  favorable  cases  led  to  experi- 
ments with  low  temperatures.  With  this  idea  in  mind,  the  slides 
sown  with  spores  were  placed  in  an  ice  box  of  the  usual  construc- 
tion. A Richard’s  self -registering  thermometer  was  also  placed 
in  the  ice  box  so  that  a complete  record  could  be  had  of  the 
temperatures  to  which  the  spores  were  exposed.  In  the  case  of 
the  controls  at  room  temperature,  the  stands  holding  the  slides 
sown  with  spores  were  placed  on  a wet  earthen  plate  and  a small 
bell  glass  placed  over  them  to  prevent  rapid  evaporation.  The 
effect  of  darkness  was  also  tested  by  placing  similar  cultures  in 
th*e  dark  room.  The  temperature  of  the  dark  room  was  noted 
when  the  experiment  was  started  and  stopped  and  the  average 
taken.  The  cultures  at  room  temperature,  not  in  the  dark  room, 
were  kept  in  a greenhouse  where  another  self-registering  ther- 
mometer recorded  the  temperature.  Here  also  the  temperature 
was  noted  when  the  experiment  was  started  and  stopped.  For 
convenience  in  tabulating,  the  average  of  the  two  extremes  was 
taken  as  the  prevailing  temperature. 

Cultures  in  the  ordinary  Yan  Tieghem  cell  were  also  used.  The 
cell  was  partially  filled  with  tap-water  and  a hanging  drop  made 
containing  the  spores.  Vaseline  was  used  to  prever'L  evaporation 
and  to  hold  the  cover  glass  in  place.  The  cells  were  laid  on  a 
stand  as  described  above  for  the  slides.  Watch  crystals  were  used 
when  it  was  desired  to  secure  large  quantities  of  spores  in  differ- 
ent stages  of  germination. 


Results  of  Germination  Experiments 

My  earliest  experiments  in  germination  of  the  spores  had  the 
double  aim  of  providing  material  for  the  cytological  study  of  the 
processes  of  nuclear  and  zoospore  formation  in  the  germination  of 
the  conidia  and  of  obtaining  a reliable  method  for  germination  of 
the  conidia  for  infection  experiments  in  determining  whether 


34 


Wisconsin  Experiment  Station. 


physiological  species  are  to  be  found  in  the  group.  As  noted 
earlier,  it  was  found  difficult  to  obtain  germination  and  a long 
series  of  one  hundred  experiments  was  made,  testing  the  general 
questions  as  to  the  effect  of  age  and  maturity  of  the  spores;  the 
weather  conditions  under  which  they  were  collected ; possible  in- 
fluence of  rain  water,  dew,  tap  water  and  distilled  water;  and 
the  effect  of  gathering  the  spores  at  different  times  of  the  day. 
Attention  was  turned  to  the  possible  effect  of  artificial  media  and 
a number  of  attempts  were  made  to  germinate  the  spores  of 
Cystopus  in  artificial  media,  these  experiments  being  made  in  a 
greenhouse  the  temperature  of  which  varied  from  about  33°  C. 
in  the  day  time  to  22°  C.  at  night.  First  ordinary  nutrient  agar 
was  tried  (3  gr.  meat  extract,  10  gr.  agar,  3 gr.  salt,  1000  cc. 
water).  The  conidia  were  sown  on  this  agar  in  petri  dishes  and 
kept  under  observation  for  twentyfour  hours,  but  no  germination 
resulted  in  any  of  the  ten  experiments  tried.  Beef  bouillon  (3  gr. 
meat  extract,  3 gr.  salt,  1000  cc.  water)  was  tried  in  two  experi- 
ments, but  gave  no  germination.  Following  this,  some  special 
media  were  tested.  Ten  experiments  were  tried  with  lima  bean 
agar,  as  prepared  by  Clinton  (1908:904)  for  growing  Phytoph- 
thora,  and  eight  experiments  with  his  pumpkin  agar ; none  of  the 
cultures  showed  any  signs  of  germination  at  the  end  of  twenty- 
four  hours.  Various  other  forms  of  artificial  media  were  tested, 
including  mustard  leaf  decoction,  four  trials;  corn  meal  agar, 
five  trials ; a two  per  cent  sugar  solution,  six  trials.  The  cultures 
were  kept  under  observation  in  each  case  for  twenty-four  hours. 
In  no  case  did  germination  result  either  by  germ  tubes  or  by 
zoospores.  The  above  experiments  were  made  during  July  and 
August,  1909.  The  conidia  used  were  from  Cystopus  candidus, 
C.  bliti,  C.  cubicus  and  C.  portulacae.  They  were  collected  at 
various  times  of  the  day  ranging  from  seven  o’clock  in  the  morn- 
ing until  seven  in  the  evening,  the  great  majority  being  gathered 
about  eight  o ’clock  in  the  jnorning.  The  infected  leaves  were  cut 
off  from  the  host  plants  and  carried  to  the  laboratory,  where  the 
material  was  used  immediately.  A number  of  times  the  infected 
leaves  were  immediately  placed  in  a damp  chamber  after  they 
were  removed  from  the  host  plant.  A large  number  of  tests  were 
made  with  both  young  and  old  conidia,  before  and  after  the  epi- 
dermis of  the  pustule  had  ruptured. 

These  conidia  were,  of  course,  also  tested  in  water  on  a slide  or 
in  a hanging  drop  in  a so-called  Van  Tieghem  cell.  The  slides 


Experiments  on  Spore  Germination. 


35 


were  placed  on  a small  stand  on  a wet  earthen  plate  under  a bell 
jar  in  the  greenhouse.  In  all,  fifty-four  trials  were  thus  made  in 
water  and  in  no  case  was  germination  observed.  Each  experiment 
lasted  for  twenty-four  hours  and  several  observations  were  made 
during  that  time. 

The  effect  of  chilling  was  next  tried,  both  on  various  nutrient 
media  and  in  tap  water.  No  germination  was  obtained  in  this 
way  when  using  nutrient  media  but  with  water,  germination  was 
secured.  Thus,  when  four  slides  were  prepared  as  before  and 
placed  on  a metal  stand  in  an  ice  box,  the  conidia  had  germinated 
by  the  production  of  zoospores  in  the  course  of  1%  hours.  A 
large  number  of  cultures  were  subsequently  made  by  this  method 
to  secure  material  for  cytological  study  and  with  controls  kept  at 
room  temperature  to  show  the  exact  value  of  the  chilling  in  influ- 
encing germination.  The  results  are  strikingly  uniform  and  in 
strong  contrast  with  those  obtained  before  without  chilling. 

Summary  of  Table  I 

The  first  experiment  with  chilling  in  germinating  the  conidia  of 
Cystopus  was  made  on  August  10,  1909.  From  that  date  up 
until  April  9,  1910,  experiments  were  carried  on  as  indicated  in 
the  table.  In  all,  197  experiments  were  made,  giving  germina- 
tion in  147  cases.  From  the  number  of  those  which  showed  no 
germination  should  undoubtedly  be  subtracted  the  results  ob- 
tained on  August  12  and  15  with  conidia  of  Cystopus  portulacae. 
The  plant  from  which  the  conidia  were  obtained  had  been  dug  up, 
potted  and  taken  to  the  green  house  July  25.  It  died  in  the  course 
of  ten  days  and  the  vitality  of  the  conidia  may  have  been  reduced 
by  its  condition  on  August  2.  If  these  experiments  are  omitted 
the  number  of  failures  to  germinate  is  reduced  by  ten  and  we 
should  have  about  78  per  cent  of  the  experiments  showing  germi- 
nation and  about  22  per  cent  negative. 

As  shown  in  the  table  the  conidia  of  four  different  species  of 
Cystopus  were  used  in  these  experiments,  including  Cystopus 
candidus  78  trials,  Cystopus  cubicus  60,  Cystopus  bliti  45,  and 
Cystopus  portulacae  14.  Until  October  19  the  conidia  were  taken 
from  live  plants  growing  out  of  doors  and  used  immediately.  The 
terms  “old”  and  “young”  as  used  in  the  table  indicate  only  the 
approximate  age  of  the  conidia.  The  conidia  "were  called  young, 
although  the  epidermis  of  the  pustules  had  ruptured,  as  long  as 


36 


Wisconsin  Experiment  Station, 


Table  I. — Effect  of  Lowering  Temperature  on  the  Germination 
of  the  Conidia  of  Certain  Species  of  Cystopus. 


Date. 

No.  of 
cultures. 

Species  tested. 

Age  of 
spores. 

Period 

friger 

Hrs. 

OF  RE- 
ATION. 

Temp. 

l 

Re-  1 
suit. 

Out-door 

tempera- 

ture. 

Minimum. 

Aug'.  10. . 

4 

Cystopus  bliti 

Young  . . 

1.5 

21 

+ 

17 

Aug.  11.. 

4 

C.  candidus 

Young  . . 

2 

21 

+ 

15 

Aug-.  12  : 

2 

C.  candidus 

Young  . . 

1 

21 

+ 

19 

Aug.  12. . 

2 

C.  bliti 

Young  . . 

1 

21 

+ 

19 

Aug.  12.. 

2 

C.  candidus 

Young  . . 

1.5 

21 

+ 

19 

Aug.  12.. 

2 

C.  bliti 

Young  . . 

2 

21 

+ 

19 

Aug.  12 

2 

C.  bliti 

Old 

2 

21 

+ 

19 

Aug.  12. . 

4 

C.  portulacae 

Young . . 

2.5 

21 

19 

Aug.  13. . 

4 

C.  bliti 

Young  . . 

2.66 

20 

+ 

18 

6 

Old 

3.5 

14 

2i 

Aug.  16 

2+ 

Young . . 

1.5 

18 

Aug,  16.: 

3 

C.  candidus 

Young  . . 

2 

is 

f 

18 

Aug.  18.. 

8+ 

C.  candidus 

Young . . 

2 

18 

+ 

17 

Aug.  20 

3+ 

Young . . 

2.5 

+ 

19 

Aug.  24. . 

4 

C.  candidus 

Young . . 

11.75 

18 

12 

Aug.  23.. 

4+ 

C.  candidus 

Old 

2 

17 



17 

Aug.  23.. 

4+ 

C,  candidus 

Young . . 

24 

17 

+ 

17 

Aug.  23.. 

5 

C.  bliti 

Young . . 

2.5 

17 

+ 

17 

Aug.  23.. 

4 

C.  bliti 

Young . . 

4.5 

17 

+ 

17 

Sept.  25. . 

2 

C.  candidus 

Young . . 

24 

7 



6 

Sept,  27.. 

3 

C.  candidus 

Young . . 

7 

6 

+ 

6 

Sept.  27.. 

1* 

C.  candidus 

Young . . 

7 

6 

+ 

6 

Sept.  27.. 

1* 

C.  candidus 

Youug . . 

24 

6 

+ 

6 

Oct.  1... 

4* 

C.  cubicus 

Young . . 

36.5 

11 

+ 

6 

Oct.  1... 

3* 

C.  cubicus 

Young . . 

28 

10 



6 

Oct,  28... 

2 

C.  candidus 

Young . . 

4.5 

8 



8 

Oct,  28... 

1* 

C.  candidus 

Young  . . 

4.5 

8 

+ 

8 

Sept.  23.. 

2* 

C.  cubicus 

Young  . . 

5.75 

8 

+ 

8 

Sept.  23.. 

1 + 

C.  bliti 

Young  . . 

4.5 

8 

+ 

8 

Oct.  4... 

1 + 

C.  bliti 

Young  . . 

3 

8 

+ 

6 

Oct,  4... 

1* 

C.  bliti 

Young . . 

3 

8 

+ 

6 

Oct.  4... 

,3* 

C.  cubicus 

Young  . . 

4 

9 

+ 

6 

Oct.  9. . . 

2* 

C.  candidus 

Young  . . 

3.5 

10 

+ 

13 

Oct.  9.. 

Z* 

C.  candidus 

Young  . . 

16 

10 

+ 

13 

Oct.  14... 

2+ 

C.  bliti 

Young  . . 

15 

13 

+ 

— 2 

Oct.  14... 

3* 

C.  bliti 

Young  . . 

2.66 

12 

+ 

2 

Oct,  28. . . 

2 

C.  candidus 

Young  . . 

7.5 

12 

+ 

— 4 

Oct.  8... 

2+ 

C.  bliti 

Young  . . 

6 

11 



11 

Oct,  19... 

4 

C.  cubicus 

Young  . . 

6.25 

10 

__ 

1 

Oct.  10. . . 

3 

C enhiens  .... 

Young  . . 

3 

10 

+ 

( >,‘t.  10. . . 

3 

C bliti 

Young  . . 

3 

10 

.T an . 24. 

4 

C.  candidus 

Young  . . 

47 

11 

Jan.  25. 

3 

C.  candidus. . . . 

Young  . . 

10 

10 

4 

Jan.  26. 

4 

C.  c.a.ndidns 

Young  . . 

22 

11 

.1  an.  26. 

3 

C rand  id  ns 

Young  . . 

12 

11 

-+- 

Aug.  25" 

1 

C.  bliti 

Young  . . 

5.5 

18 

4 

20 

Aug.  28. . 

4 

C.  candidus 

Young  . . 

6 

14 

4 

16 

Aug.  30.. 

4 

C.  portulacae 

Young  . . 

3 

15 

4 

11  ' 

Aug.  31.. 

3+ 

C.  bliti 

Young  . . 

4.25 

11 

4 

8 

Aug.  31.. 

1 

C.  cubicus 

Young  . . 

18.5 

11 

4 

8 

Sept,  2.. 

1 + 

C.  cubicus 

Young  . . 

2.12 

10 

— 

7 

Sept.  4.. 

4 

C.  cubicus 

Young  . . 

10.75 

10 

4- 

12 

Sept.  4.. 

o 

C.  bliti  

Old 

2.5 

-10 

+ 

12 

Sept.  4.. 

1 

C.  candidus 

Young . . 

2.5 

10 

12 

Sept.  6.. 

1 

C.  cubicus  . 

Old 

5 25 

8 

4 

9 

Sept.  6.. 

1 

C.  cubicus 

Old..  .. 

5.25 

8 

9 

Sept.  7 . . 

2 

C.  cubicus 

Old 

6.5 

9 

4 

9 

Sept.  10.. 

4+ 

C.  cubicus 

Young  . . 

4.25 

11 

+ 

13 

Sept.  10.. 

1 

O.  candidus 

Young  . . 

7 

11 

4 

13 

Sept.  13. . 

3 

C.  cubicus 

Young  . . 

2.33 

8 

4 

18 

Sept.  14. . 

4 

O.  cubicus 

Young  . . 

2.75 

8 

4 

It 

Sept,  14. . 

4 

C.  cubicus 

Young  . . 

3 

9 

4 

1 + 

Sept.  16. . 

1* 

O.  Cubicus 

Young  . . 

4.87 

10 

4 

9 

Sept.  20. . 

1* 

C.  cubicus 

Young  . . 

5.5 

4 

15 

Sept.  21 . . 

4+ 

C.  candidus 

! Young . . 

9.25 

12 

i 4 

16 

* Experiments  carried  on  in  watch  crystals. 

t Pieces  of  leaves,  on  which  there  vere  pastilles,  were  laid  on  the  slide. 


Experiments  on  Spore  Germination. 


37 


Table  I.  Continued. — Effect  of  Lowering  Temperature  on  the 
Germination  of  the  Conidia  of  Certain  Species  of  Cystopus. 


Date. 

No.  of 
cultures. 

Species  tested. 

Age  of 
spores. 

Period 

frigei 

Hrs. 

> OF  RE- 
LATION. 

Temp. 

Re- 

sult. 

Out-door 

tempera- 

ture. 

Minimum. 

Sept.  21.. 

1 

C.  candidus 

Young . . 

23 

14 

+ 

16 

Sept.  21 . . 

1* 

C.  candidus 

Young 

23 

14 

+ 

16 

Sept.  21.. 

1 

C.  cubicus 

Young  .. 

4.87 

12 

+ 

16 

Sept.  21 . . 

3 

C.  blip 

Young . . 

29 

1.4 

+ 

16 

Sept.  22.. 

4* 

C.  cubicus 

Young . . 

6 

11 

+ 

n 

Sept.  24. . 

3* 

C.  cubicus 

Young . . 

3.75 

8 

+ 

7 

Sept.  25. . 

4 

C.  cubicus 

Old 

23 

y 

Q 

Sept.  25. . 

2* 

C.  cubicus 

^ Young  . . 

3.25 

7 

+ 

6 

^Experiments  carried  on  in  watch  crystals. 

a considerable  number  of  spores  remained  in  the  pustules;  and 
old  after  the  pustules  were  nearly  empty.  The  conidia  were  either 
taken  out  of  the  pustule  and  placed  in  a drop  of  water  or  small 
pieces  of  leaves  with  pustules  were  laid  in  a drop  of  water.  If 
the  pustules  on  the  pieces  of  leaves  were  not  already  open  when 
they  were  laid  in  the  drop  of  water  the  epidermis  was  broken 
with  a needle. 

An  ordinary  ice  box  was  used  and  in  it  was  kept  a self  regis- 
tering thermometer.  By  referring  to  the  table  it  can  be  seen  that 
the  temperatures  from  August  10  to  August  25  ranged  from  15° 
to  21°  C.  The  temperature  was  usually  above  18°  C.  The  ice  box 
was  kept  in  a rather  warm  room  adjoining  the  green  house,  and  it 
was  also  used  for  other  purposes  so  that  the  doors  were  opened 
and  closed  quite  often.  The  temperature  curve  was  very  irregu- 
lar. There  were  fluctuations  of  10°  C.  in  five  hours  in  some  cases 
although  usually  it  was  less.  Since  the  fluctuations  were  too 
numerous  to  explain  in  connection  with  each  test,  the  average  of 
me  maximum  and  minimum  temperature  Has  Deen  taRen  as  the 
prevailing  temperature  and  is  that  recorded  in  the  table.  This, 
in  some  experiments  and  especially  in  those  before  August  13, 
does  not  give  the  correct  temperature  conditions.  In  the  tests 
after  August  13,  there  was  much  less  variation  and  the  average 
of  the  two  extremes  is  much  nearer  the  prevailing  temperature 
condition.  The  exact  temperatures  during  two  tests  are  given  in 
detail  to  show  more  clearly  the  existing  conditions.  For  example, 
the  temperature  varied  as  follows  during  the  experiment  on 
August  25 : The  test  was  started  at  9 :15  a.  m.  with  a temperature 
of  20°  C.  The  temperature  remained  constant  until  10  o’clock. 
Thirty  minutes  later  the  temperature  was  21°  C.  At  11  o’clock 


38 


Wisconsin  Experiment  Station. 


it  was  19°  C.  where  it  remained  until  12  o’clock  noon.  At  1 
o’clock  it  was  down  to  14°  C.;  at  2 o’clock  it  was  13°  C.  and  at 
2 :45  p.  m.  it  again  rose  to  14°  C.  During  5 1-2  hours,  the  tem- 
perature varied  8°  C.  After  August  25,  the  temperature  of  the 
ice  box  was  much  more  constant  and  often  fluctuated  only  one 
degree  during  the  time  of  an  experiment.  The  period  of  refrig- 
eration on  September  2 started  at  9 :30  a.  m.  with  a temperature 
of  10%°  C.  and  stopped  at  2 :35  p.  m.  with  a temperature  of  11° 
C.  During  the  five  hours  of  refrigeration,  the  temperature  only 
varied  y2  degree. 

It  is  to  be  noted  also  that  the  temperature  grew  gradually  lower 
until  October  9.  This  was  due  to  the  fact  that  before  this  time, 
no  artificial  heat  of  any  consequence  was  used  in  the  green  house ; 
while  after  this  date  it  was  heated.  For  comparison  with  the 
temperature  in  the  ice  box,  the  minimum  out  door  temperature  is 
given  for  each  day  on  which  an  experiment  was  made,  as  pub- 
lished by  the  local  weather  bureau  of  Madison  in  their  monthly 
meteorological  summary.  It  will  be  seen  that  the  temperatures  in 
the  ice  box  varied  two  degrees  or  less  from  the  minima  out  doors 
in  72  per  cent  of  the  tests  until  about  October  14.  This  suggests 
that  germination  can  readily  take  place  at  temperatures  equal  to 
or  varying  two  degrees  or  less  from  the  minima  for  the  outdoors. 
The  length  of  time  required  for  germination  varied  from  one  to 
thirtysix  hours.  The  one  hour  period  required  for  germination 
was  on  August  12  and  the  thirty-six  hour  period  was  on  October 
10.  All  of  the  experiments  that  required  an  unusual  length  of 
time  for  germination  were  examined  from  one  to  six  times  before 
the  final  observation  was  made.  Water  was  added  to  replace  the 
amount  that  evaporated.  It  should  be  said,  however,  that  the 
usual  period  required  for  germination  in  the  majority  of  the 
cases  was  less  than  six  hours.  From  August  10  to  31  the  average 
length  of  the  period  of  refrigeration  necessary  to  produce  germi- 
nation was  about  3y2  hours.  The  longest  period  was  18 y2  and 
the  shortest  period,  one  hour. 

During  the  month  of  September  experiments  were  made  on 
twentyone  different  days,  one  more  than  in  August,  and  the 
average  length  of  the  period  of  refrigeration  was  about  7y2 
hours;  here  the  longest  period  was  twentynine  hours  and  the 
shortest  period  two  hours  and  five  minutes.  In  October,  experi- 
ments were  made  on  nine  different  days.  The  average  of  the 
periods  of  refrigeration  used  where  germination  resulted  was  nine 


Experiments  on  Spore  Germination. 


39 


hours,  the  longest  period  being  S6y2  hours,  and  the  shortest  two 
hours  and  forty  minutes.  From  these  facts  it  is  strongly  sug- 
gested that  the  period  of  refrigeration  is  longer  in  the  fall  than  in 
the  summer  as  has  already  been  pointed  out  by  Zalewski 
(1883:215).  No  germination  experiments  were  carried  on  from 
October  28  to  January  24,  1910.  However,  on  January  24,  25,  26, 
1910,  fourteen  trials  were  made  in  which  six  germinations 
occurred.  The  time  required  was  ten  hours  in  the  trials  on  Janu- 
ary 24  and  twelve  houTs  in  the  three  successive  tests  on  the  follow- 
ing days.  Although  only  a small  number  of  tests  were  made  dur- 
ing the  month  of  January,  it  was  quite  evident  that  the  conidia 
responded  differently  at  this  time  than  in  the  summer.  In  the 
tests  made  in  January  the  zoospores  lost  their  motility  in  less  than 
one  hour  and  developed  long  germ  tubes.  In  none  of  the  tests 
made  before  that  time  had  germ  tubes  been  seen.  The  different 
behavior  of  the  conidia  in  the  late  fall  and  winter  as  compared 
with  spring  and  summer,  are  attributed  to  the  loss  of  vitality 
of  the  host  and  fungus  or  to  the  improper  maturing  of  the 
spores. 


Table  II. — Effect  of  Lowering  Temperature  in  Germination  of 
Conidia  of  Certain  Species  of  Cystopus,  With  Controls  at 
Room  Temperature. 


Date. 

Species  tested. 

Experiment  at  Low  Tem- 
perature. 

1 

Controls  at  Room  Tem- 
perature. 

No. 

cul- 

tures. 

Period  of  re- 
frigeration. 

Re- 

sults. 

No. 

cul- 

tures. 

Time  con- 
tinued. 

Re- 

sults. 

Hrs. 

Temp. 

Hrs. 

Temp. 

Aug1.  11. . 

Cystopus  bliti. . 

4 

1.5 

21 

+ 

4 

1.5 

25 

Aug.  18. . 

C.  bliti 

8 

2 

18 

+ 

8 

2 

’ 27 

Au g.  20.. 

C.  bliti 

3 

2.5 

19 

+ 

3 

2.5 

27 

Aug.  21.. 

C.  bliti 

4 

10 

18 

4 

10 

25.5 

Aug.  23. . 

C.  bliti 

9 

7.3 

17 

+ ' 

9 

7.3 

27 

Aug.  16. . 

C.  candidus 

5 

1.6 

18 

+ 

5 

2 

28.5 

1 A 

Sep.  15.. 

C.  bliti 

2 

5.5 

10 

+ 

2 

5.5 

22 

Sep.  25.. 

C.  candidus 

2 

24 

8 

2 

24 

22 

Oct.  .9.. 

C.  candidus 

2* 

5.5 

11 

+ 

2* 

23.75 

22 

Oct.  9.. 

C.  candidus 

2 

28 

10 

+ 

2 

28.5 

21 

1 = 

1 

| p* 

C.  bliti 

4 

2.6 

20 

+ 

4 

14 

27 

— 

*Conidia  placed  on  watch  crystals,  instead  of  slides. 


Summary  of  Table  II 

As  noted,  no  control  experiments  were  kept  in  connection  with 
the  trials  reported  in  Table  I.  The  conidia  were  germinated  as 
material  for  cytological  study  which  will  be  reported  on  later.  A 
second  set  of  similar  experiments  (Table  II)  with  controls,  was 


40  Wisconsin  Experiment  Station. 

carried  on  to  demonstrate  beyond  question  that  chilling  is  neces 
sary  for  abundant  germination.  In  this  series  of  forty  five  cul- 
tures subjected  to  low  temperatures,  six  failed  to  germinate. 
That  is,  about  85  per  cent  of  the  tests  gave  germination.  In  the 
controls,  no  germination  was  observed.  Thq  temperature  during 
the  period  of  refrigeration  was  very  high  for  ice-box  temperature 
in  most  of  the  experiments,  due  to  the  conditions  explained  in  the 
summary  of  Table  I. 

In  all  of  these  experiments  the  conidia  were  placed  in  tap  water 
on  the  slides.  In  order  to  prevent  too  rapid  evaporation  in  the 
trials  at  room  temperature,  the  slides  were  laid  on  a metal  stand 
placed  on  a wet  plate  under  a small  *bell.  jar.  The  final  observa- 
tions were  made  in  both  sets  of  experiments  at  the  same  time  and 
the  results  recorded.  The  average  room  temperature  at  which  the 
experiments  were  made  varied  from  21  to  28.°  C.,  as  can  be  seen 
in  Table  II ; while  the  temperature  during  the  period  of  refrig- 
eration varied  from  8°  to  21°  C.  Otherwise  the  conditions  were 
the  same  in  both  sets  of  experiments.  These  results  show  that  a 
slight  lowering  of  the  temperature  stimulates  the  conidia  of 
Cystopus  to  germinate  with  the  production  of  zoospores. 


Table  111.— Effect  of  Light  on  the  Germination  of  the  Conidia  of 
Certain  Species  of  Cystopus. 


Controls  Chilled. 

Culture 

at  High  Temper- 
atures. 

Period  of 

Period  in 

Period 

No. 

refrig’ra’n. 

dark 

room. 

in  light. 

Date. 

of 

cul- 

Species tested. 

c/5 

• 

c/5 

Ji 

c/5 

a 

C/3 

tures 

|.  3 

s 

c n 

2 

I 

C/3 

3 

S 

9 

C/3 

1 5 

H 

(S 

& 

H 

M 

K 

H 

Aug.  11. 

4 

Cvrstopus  Candidas 

: 2 

21 

-f  , 

8 75 

26 

Aug.  13. . 

4 

c‘.  bliti 

2.66 

20 

+ 

U. 

27 



14. 

27 

— 

Aug.  20. . 

3* 

C.  bliti 

2.5 

18 

4- 

1.5 

28 

— 

2.5 

27 

— 

Aug.  23. . 

4* 

C.  bliti 

9. 

17 

+ 

4. 

27 

— 

7.33 

27 

— 

Sept.  15.. 

2 

C.  bliti 

5.5 

10 

+ 

5.5 

27 

— 

5.5 

22 

— 

Sept.  25.. 

2 

C.  candidus 

24. 

8 

24. 

28 

— 

24. 

22 

— 

Oct.  9. . . . 

24 

C.  candidus 

5.5 

11 

+ 

6. 

27.5 

— 

23.75 

22 

* Pieces  of  leaves  on  which  there  were  pustules,  were  laid  od  the  slide, 
•('Experiments  carried  on  in  watch  crystals. 

Summary  of  Table  III 

The  possible  effect  of  light  on  the  germination  of  the  conidia  of 
Cystopus  was  also  tested.  Controls  were  kept  to  ascertain  the 
viability  of  the  conidia  and  were  chilled  as  described  above. 
Twenty-one  cultures  were  exposed  to  light  and  seventeen  were 


Experiments  on  Spore  (termination. 


41 


kept  in  darkness,  in  each  case  without  chilling.  All  failed  to 
germinate.  The  controls  all  germinated  except  two.  These 
results  are  shown  in  Table  III.  All  the  experiments  were  carried 
on  simultaneously  and  all  the  conditions  were  the  same  except 
that  no  bell  jars  were  used  to  cover  the  controls  while  bell  jars 
were  used  in  the  experiments  in  the  light  and  in  the  dark.  The 
conidia  were  taken  from  freshly  matured  pustules  and  placed  in 
a drop  of  water  on  slides  that  were  well  cleaned.  One  series  of 
cultures  was  kept  in  the  diffused  light  of  an  ordinary  laboratory 
where  no  direct  sunlight  fell  upon  them;  the  other  series  was 
kept  in  a dark  room.  The  high  temperatures,  from  August 
11  to  25,  have  been  explained  in  connection  with  Table  I.  The 
question  might  naturally  be  raised  as  to  whether  germination  of 
the  conidia  in  the  ice  box  were  not  due  to  the  dark  moisture 
saturated  atmosphere  of  the  ice  box  rather  than  to  the  low  tem- 
perature. These  experiments  answer  this  question.  The  series  of 
seventeen  cultures  kept  in  the  dark  room  were  in  a saturated 
atmosphere  the  same  as  the  controls  in  the  ice  box.  The  only  dif- 
ference was  in  the  temperature  which  varied  from  26°  to  28°  C. 
in  the  dark  room  and  from  8°  to  21°  C.  in  the  ice  box.  The  ice 
box  cultures  all  germinated  except  two ; while  none  of  the  seven- 
teen cultures  in  the  dark  room  germinated.  From  these  results  it 
is  clear  that  light  is  not  a determining  factor  in  germination.  It 
is  also  clear  that  a saturated  atmosphere  at  high  temperatures 
will  not  cause  the  germination  of  the  conidia  of  Cystopus. 

Table  I Y.— Effect  of  Using  Still  Lower  Temperatures  in  Germina- 
tion of  Conidia  of  Certain  Species  of  Cystopus. 


Date. 

No.  of 
cul- 
tures 

Species  tested. 

Age  of 
spores . 

Period  of  refriger- 
ation. 

Re- 

sults. 

Out-door 

tempera- 

ture. 

minimum. 

Hours. 

Temp. 

Sept.  9.. 

1 

Cystopus  bliti 

Young 

1.16 

§ 

15 

Sept.  9.. 

2 

C.  cubicus 

Young 

8 

§ 

+ 

15 

Sept.  9.. 

1 

C.  candidus 

Young 

8 

§ 

+ 

15 

Sept.  13.. 

8* 

C.  cubicus 

Young,. . . 

2.5 

—1 

— 

18 

Sept.  8.. 

2 

C.  cubicus 

Young.. . . 

4.5 

§ 

-+- 

12 

Sept.  4.. 

1+ 

C.  bliti 

Young 

19.5 

§ 

+ 

14 

Sept.  27.. 

1 + 

C.  cubicus 

Young... . 

27 

§ 

— 

6 

Sept.  27.. 

It 

C.  cubicus 

Young... . 

27 

§ 

+ 

6 

Oct.  4.. 

1 

C.  cubicus 

Old 

10.75 

§ 

4- 

6 

Oct.  4.. 

1 

C.  bliti 

Young 

10.75 

§ 

+ 

6 

Oct.  12.. 

3 

C.  bliti* 

Young.,. . 

3 

10 

— 

-4 

Oct.  12.. 

3 

C.  cubicus? 

Young 

3 

10 

+ 

—4 

Oct  16 

2 

O e.iihimist 

Old 

3 

12 

+ 

0 

Oct.  16.. 

4 

C.  bliti? 

Old 

3 

12 

+ 

0 

*This  experiment  was  made  with  a Van  Tieghem  cell  and  hanging  drop. 
tPiec^s  of  leaves  with  pustules  in  water  on  watch  crystal. 

^Conidia  taken  from  frozen  leaves. 

§The  slides  in  these  experiments  were  laid  on  a block  of  ice  in  the  ice  box. 


42 


Wisconsin  Experiment  Station. 


Summary  of  Table  IV 

ihe  effects  of  lower  temperatures  than  those  ordinarily  obtained 
in  the  ice  box  were  also  tried.  The  results  of  these  experiments 
are  shown  in  Table  IV.  The  slides  were  laid  on  blocks  of  ice, 
except  in  the  case  of  the  experiments  on  September  13  and 
October  12  and  16,  which  are  further  described  below.  Twelve 
cultures  were  made.  In  nine,  the  spores  germinated  while  in 
three,  there  was  no  germination.  These  results  are  somewhat 
at  variance  with  DeBary,  who  found  the  minimum  to  be  5°  C. 
There  can  be  no  doubt  from  the  above  results  that  the  minimum 
for  germination  is  very  near  0°  C. 

To  test  the  effect  of  still  lower  temperatures,  three  Van 
salt  giving  a temperature  of  1°  C.  At  the  end  of  thirty  minutes 
no  germination  had  occurred.  These  slides  were  allowed  to 
remain  in  the  laboratory  at  28°  C.  for  ten  hours  after  being  re- 
moved from  the  freezing  mixture,  but  no  germination  resulted. 
This  indicates  that  a change  from  high  to  low  and  then  back  to 
high  does  not  lead  to  germination. 

The  effect  of  frost  on  the  eonidia  of  Cystopus  outdoors  was  also 
tested.  The  eonidia  were  collected  October  12  and  16  from  frozen 
leaves  which  had  been  allowed  to  thaw  out  in  the  laboratory. 
Twelve  cultures  were  kept  at  10°  to  12°  C.  for  three  hours.  Nine 
of  the  twelve  cultures  germinated  and  three  did  not,  indicating 
that  the  eonidia  were  not  killed  by  a frost. 

Summary  of  Table  V 

In  view  of  the  fact  that  germination  was  obtained  in  the  ice 
box  at  temperatures  above  20°  C.  in  some  cases,  it  was  thought 
advisable  to  make  a further  study  of  the  relation  of  temperature 
to  spore  germination  at  room  temperatures.  During  the  latter 
part  of  March  and  first  part  of  April,  1910,  further  experiments 
were  made  to  determine  whether  the  eonidia  of  Cystopus  would 
germinate  at  room  temperatures.  As  previously  described,  the 
experiments  with  cultures  at  green  house  temperatures  in  the 
summer  of  1909  had  given  oply  a low  percentage  of  germination. 
In  fact  germination  was  observed  in  only  one  or  two  cases.  In 
the  new  series  of  experiments,  seventythree  cultures  were  made 
from  March  27  to  April  8,  at  temperatures  varying  from  17%°  to 
25°  C.  Controls  were  kept  at  ice  box  temperatures  in  cases 


Table  V — Further  Experiments  on  Germination  of  Conidia  of  Cystopus  at  Room  Temperature  From  March  27  to  April  8. 


Experiments  on  Spore  Germination.  43 


Result. 

* * 

-H-+ 1 1 1 

1 1 +++++++++++  1 +++*!+* 

* * *■ 

1 l++x  l 

rv 

c3 

: : : : ^ : 

: : : : : >,  : ; ^ ^ ’.  : -.  >s  ; : 

; ; ^ ; ; ; 

w 

II!  :*o  ; 

: : : : i'o-'  i :t3  : i i'S  : : . : 

. .rQ  • • • 

CM  CM  CM  CO  irt  lO  co 


CO  CO  CO*  CM  CM  CM  CM 


UO  HO  m ^ CM  CM  - 


<D  <d  d)  Cu  Th  O 0 <£»  d)  ^ ^ ^ Q)  Th 


. . £ g C C c3- 
OOSOOD' 


0)  O q O a, 


0)  0513  05 

35  .ti  O o ^ °-~35 


cd-»cd-cdcd*cd*****cdcO 
rCrt3rdrO  f-  wT3rc!  *-t'OrO  ^T3  ft  f-n3  ^ 'C T3 T3 'O  5-1  ’~rr3rr3r&  «rwT3rO'C! 
05a5a5aJt3S05ajr3a)ai3o5Si3Qji3a)a;Q5ai<D!3pia5ajajSa5a5a50) 

cocOrfcdcccOcdcScdCjcdcS^cdOTcdcicOcdcdJScScOcdcdcOcOeacdcdcdrt 

rtk2p5'^'^5'cl  ^ s3>5  & ^ ^ 

ooaZj.ZZ  x £ x x x ix/Zxx  x ^xxx  x 


x x xxl?Z  x x £ x : 


o-S 

ra'cs 

0^3 


lO  iO  lO  UO 
^^COCOC5CMCM05CMCMCMCMCMCMi-Ii 


It-itHi-iCMCMCMCMCMCMi 


IH 


CO  CO  CO  CO  l>- 1>»  t—  l>- 


irs  10 10  >0 10 10 


a)  ^ co 
ha  55  &j 

<!§5 


05c3>oiciO!MeaocofO'^'^'^-^vftif5ioif5imraimf5coa5cococceciftift>i5ir5 


si»*«!»iiiima!mi/iiB 

ODSPS33333S 

^rt3rcn3T3rcrOrorbrcrc;rbro,c!rw,ararorc' 

E^SSScdSScoSeScScdcOcdcScOcdcd 

CCOOOOOOOOOOOOOOOOOO 


•yjtflcfitwt/itn'yjasMi/itnt/lajMMcfitncntKQD 

33:3:3:33322322233135:332 

^T3232!r3rOr3r3’25r3r3rOr3r3rO,3;3r3;3r3 

,FH  *”  "|  jfi ,rH  ,rH  'S  'S  S S 'S  'S  ^3  S S S £ 

scs  C'O  ccaaccfl 

cdcdcdcOcdcdcdcdcdcdcdcO 
OOOOOOOOOOOO 


OOOOOOOQOOOOOOOOOOOOOOOOOOOOOOOO 


i«(Mt)(NN(NNNMNNWO!NWNNHHHHNWNN' 


NNNNOOOOOOOOrtrtHHNN«NNWNN^'<Ji^'#^'#tO(ONOO 
W(M(M(M  COCOCMCOCO 

o o o o o o o o o o .7:  53  3 35  35  35  S .73 .-  53  S 32  .53 .73  53  53  53 .53  53  53  35  33 
cocedddddddojftaaaaaaQiaa  a'a  aaaaaaaaaa 
S 3 S S S S S S S S <3  <5  <1  <1  <1  <5  <1  < <J  <h  <5  <1  <3  <3  <!  <1  <3  <1  <5  <i  ■<  <3 


Ice  water  used.  XTwo  cultures  geiminated  and  two  did  not. 


44 


Wisconsin  Experiment  Station. 


where  the  temperature  of  the  room  was  about  20°  C.  The  room 
where  this  series  of  experiments  was  carried  on  was  lighted  by  a 
skylight.  It  was  steam  heated  and  had  only  one  door.  The 
temperature  during  any  one  experiment  never  varied  more  than 
four  degrees.  The  temperature  was  noted  when  the  experiment 
was  started  and  stopped  and  the  average  of  the  two  readings  are 
given  in  Table  V.  The  conidia  were  obtained  from  stock  cultures 
kept  growing  in  the  green  house  during  the  winter.  The  age  of 
the  conidia  was  determined  as  far  as  practicable  by  noting  the 
time  of  appearance  of  the  pustules  and  each  time  using  conidia 
from  the  marked  pustule.  The  plants  serving  as  the  source  of 
conidia  were  placed  on  a separate  bench  where  no  wind  could 
strike  them,  so  that  the  conidia  could  not  be  blown  away.  Care 
was  exercised  in  watering  the  plants  not  to  jar  the  infected  leaves. 
In  this  way  it  was  possible  to  obtain  conidia  from  pustules  of 
known  age.  The  spores  were  placed  in  tap  water  except  on  March 
27  and  April  4 when  ice  water  was  used.  Twenty  tests  were  made 
without  covering  the  cultures  with  bell  jars;  while  the  remaining 
fiftythree  were  kept  in  a saturated  atmosphere  obtained  by  plac- 
ing the  cultures  under  bell  jars  placed  on  a wet  earthen  plate. 
The  approximate  amount  of  evaporation  in  the  cultures  was  ob- 
served and  recorded  in  each  case.  In  this  case  of  seventythree 
tests  at  room  temperature  varying  from  17%°  to  25°  C.,  twenty- 
six  failed  to  germinate.  Controls  were  kept  at  lower  temperatures 
all  of  which  germinated  readily,  indicating  that  the  conidia 
used  were  normal.  The  time  required  for  germination  varied 
from  forty-live  minutes  to  four  hours.  The  former  being  the 
shortest  period  at  which  germination  was  observed.  Twenty 
tests  were  made  in  a non-saturated  atmosphere  and  fifty- three 
in  a saturated  atmosphere.  Nine  of  the  cultures  that  were  sub- 
jected to  ordinary  room  conditions  failed  to  germinate,  and 
eleven  of  the  cultures  that  were  in  a saturated  atmosphere  failed. 
This  suggests  that  a saturated  atmosphere  is  not  necessary  so 
long  as  the  conidia  are  in  water.  The  germination  apparently 
took  place  as  readily  in  cultures  nearly  dry  as  when  no  evapora- 
tion occurred.  It  should  be  noted  still  further  that  twenty-five 
cultures  were  made  at  temperatures  above  20°  C.  in  which 
58  per  cent  germinated.  The  remaining  forty-eight  culture  in 
the  series  were  at  temperatures  between  17%°  and  20° 
C.  or  below  and  60  per  cent  germinated.  From  these 
results  it  is  again  clear  that  temperature  is  an  important  factor 


Experiments  on  Spore  Germination. 


45 


in  the  germination  of  the  conidia  of  Cystopus.  These  results 
would  have  been  still  more  striking  had  the  temperatures  in  the 
last  series  been  nearer  the  optimum,  10°  C. 


Table — VI.  Observations  on  Outdoor  Germination  of  Cystopus 
on  Radish  and  Salsify. 


Results  observed. 

Outdoor  weather  conditions. 

Date. 

Time 
of  dav 
a.  m. 

Species  of 
plant. 

•‘■Zoospores. 

Max. 

Min. 

Preci- 

pita- 

tion. 

Character 

Present. 

Absent. 

Temp. 

Temp. 

of  day. 

Sept.  9. . 
Sept.  11.. 
Sept.  15.. 
Sept.  20.. 
Sept.  20.. 
Sept.  28.. 
Oct.  7. . 

8 

Salsify. ... 

abundant. . 

16 

8 

0 

Clear. 

8 

Salsify.. . . 
Salsify... . 

abundant. . 

26 

10 

.04 

Cloudy. 

8 

abundant . . 

20 

11 

0 

Clear. 

5 

Radish... 

abundant. . 

21 

10 

0 

Clear. 

5 

Salsify.... 
Salsify 

abundant. . 

21 

10 

0 

Clear. 

8 

abundant . . 

19 

8 

0 

Clear. 

9:15 

Salsify.... 

absent.. 

19 

6 

0 

Clear. 

Oct.  21.. 

8 

[ Salsify.... 

abundant. . 

10 

5 

.22 

Cloudy. 

Summary  of  Table  YI 

Since  it  was  found  that  the  spores  of  Cystopus  germinated 
readily  in  the  laboratory  when  chilled,  my  attention  was  directed 
to  the  possibility  that  chilling  also  favored  germination  on  the 
host  plant  out  doors  when  a dew  was  present.  Observations  were 
made  on  seven  different  days  from  September  9 to  October  21. 
The  observations  were  made  on  the  leaves  of  salsify  (Tragopogon 
porrifolius)  and  radish  (Raphanus  sativus)  which  were  badly 
infected  with  Cystopus.  The  leaves  were  gathered  from  5 to  9 :15 
a.  m.  and  were  carried  to  the  laboratory  and  examined  for  motile 
zoospores.  The  zoospores  were  found  in  every  case  except  on 
October  7.  On  this  date  the  observation  was  made  at  9 :15  a.  m. 
rather  than  at  8 :00  a,  m.,  which  was  the  usual  time.  It  should 
be  noted  that  no  observations  were  made  after  9:15  a.  m.  when 
the  leaves  were  wet.  This  should  undoubtedly  be  done  to  fully 
prove  that  germination  takes  place  only  in  the  morning  or  when 
the  temperature  is  low.  The  minimum  for  the  days  on  which  ob- 
servations were  made  varied  from  5 to  11  2-3°  C.  as  seen  from  the 
table.  The  lowest  temperature  for  the  days  in  question  came 
about  sunrise  as  is  usually  the  case.  It  is  quite  clear  from  these 
observations  that  germination  outdoors  does  take  place  at  .the 
time  of  lowest  temperature  for  the  day.  The  question  as  to 
whether  germination  takes  place  at  any  other  time  of  the  day 
will  be  further  investigated  this  coming  summer. 


46 


Wisconsin  Experiment  Station. 


Results  with  Other  Species  of  Peronosporaceae 

It  has  been  found  that  the  favorable  effect  of  chilling  is  not 
restricted  to  the  conidia  of  Cystopus,  but  is  manifest  as  well  in 
the  case  of  the  conidia  of  other  Oomycetes.  I have  incidentally 
tested  the  germination  of  the  conidia  of  Plasmopara  viticola  at 
two  different  times  and  each  time  found  that  the  conidia  germ- 
inated readily  at  a temperature  of  about  10°  C.  Pour  tests  were 
made  with  Peronospora  effusa  and  germination  resulted  in  two  to 
four  hours  when  kept  at  10°  C.  Two  trials  were  made  with  the 
conidia  of  Peronospora  parasitica  and  germination  resulted  in 
two  and  one  half  hours  at  12°  C.  The  conidia  of  Phytophthora 
infestans  also  germinated  when  chilled  for  2%  hours  at  12°  C. 
The  controls  for  each  of  the  above  experiments  failed  to  germinate 
at  room  temperature.  These  results  are  based  on  only  one  to  four 
trials  with  each  species,  but  show  that  chilling  favors  the  germ- 
ination of  conidia  of  other  Oomycetes  as  well  as  those  of  Cysto- 
pus. Further  experiments  are  in  progress  to  determine  more 
fully  the  effect  of  chilling  on  the  germination  of  the  conidia  of 
Plasmopara,  Peronospora  and  Phytophthora. 


GROWING  CYSTOPUS  IN  STOCK  CULTURES  UNDER 
GREEN  HOUSE  CONDITIONS 

It  might  naturally  be  supposed  that  so  vigorous  a parasite  as 
Cystopus  candidus  would  maintain  itself  quite  easily  under  green 
house  conditions,  but  it  was  soon  evident  that  this  is  not  the  case. 
New  infections  were  very  reluctant  to  appear  even  when  the  host 
plants  were  well  infected  in  the  beginning  and  the  old  infections 
after  a time  would  die  out.  In  order  to  maintain  through  the 
winter  vigorous  stock  cultures  several  methods  were  tried. 

I first  attempted  to  learn  the  affect  of  varying  the  light  in  the 
case  of  well  infected  plants.  In  this  experiment  ten  radish,  three 
Amaranthus  and  two  white  mustard  plants,  all  of  which  were 
well  infected  with  Cystopus  wTere  placed  on  an  isolated  bench  in 
the  green  house  where  only  diffuse  light  was  accessible.  Over 
these  plants  a large  bell  jar  was  placed,  but  free  ventilation  was 
provided  by  allowing  the  bell  jar  to  rest  on  two  bricks,  one  on 
each  side  of  the  pots  containing  the  plants.  On  September  24, 
1909,  fourteen  other  plants  for  controls  were  placed  where  they 


Experiments  on  Spore  Germination. 


47 


could  get  direct  sunlight,  also  under  similarly  ventilated  bell  jars. 
All  these  plants  were  grown  in  three  inch  pots,  and  were  four  to 
six  inches  high  and  had  four  to  eight  leaves  when  the  experiment 
was  begun.  Daily  observations  were  made  when  the  plants  were 
watered.  The  experiments  were  continued  from  September  24 
to  October  29. 

It  was  quite  evident  that  internal  growth  of  the  fungus  took 
place  both  in  the  diffuse  and  in  the  direct  light  because  hyper- 
trophy of  the  tissues  about  the  infected  areas  occurred  on  the 
leaves  of  the  radish  plants  as  well  as  the  production  of  the 
oospores  in  the  leaves  of  the  specimens  of  Amaranthus  retro - 
flexus.  At  no  time  did  new  pustules  appear  on  any  of  the  plants 
used.  It  was  thus  evident  that  Cystopus  would  grow  under  bell 
jars  in  the  green  house  both  in  direct  and  diffuse  light  but  at  no 
time  did  new  conidial  pustules  form.  Some  condition  necessary 
for  the  production  of  conidia  was  lacking. 

Further  experiments  were  made  to  learn  whether  the  fungmi 
could  be  made  to  spread  from  an  infected  to  a non-infeeted  plant 
when  in  close  contact  and  under  the  same  conditions  of  moisture, 
light,  and  temperature  described  in  connection  with  the  above 
experiment. 

On  September  27  a three  inch  pot  that  had  three  infected 
plants  in  it  was  buried  in  sand  on  a bench  in  the  sunlight. 
Around  the  infected  plants  were  seven  pots  containing  twentyone 
plants  not  infected.  A large  bell  jar  was  placed  over  the  whole 
in  such  a way  that  plenty  of  air  was  accessible.  On  October  13, 
new  infections  were  found  on  seven  new  leaves  which  had  become 
infected  during  a period  of  sixteen  days.  The  plants  were  much 
healthier  and  more  vigorous  than  the  plants  that  were  kept  in  the 
diffuse  light  in  the  above  experiment.  The  spores  were  possibly 
carried  from  plant  to  plant  by  aphids  or  by  slight  currents  of  air 
that  might  enter  under  the  bell  jar.  The  culture  was  maintained 
for  twenty  days  more  but  the  fungus  gradually  disappeared. 

In  the  following  experiment  attempts  were  made  to  produce 
new  infections  on  a large  number  of  plants  and  thus  to  retain  and 
propagate  the  conidial  stage.  Six  inch  pots,  in  which  there  were 
about  two  hundred  plants,  were  used.  A more  crowded  condition 
was  thus  produced  which  showed  itself  more  favorable  for  the 
fungus.  When  the  plants  were  eight  or  ten  days  old,  with  only 
two  cotyledons,  they  were  inoculated  with  spores  from  Cystopus 
candidus.  The  conidia  were  placed  in  water  and  sprayed  on  with 


48 


Wisconsin  Experiment  Station. 


an  atomizer.  It  was  in  this  way  easily  possible  to  inoculate  every 
cotyledon.  The  method  of  chilling  for  abundant  spore  germina- 
tion was  also  tried  on  these  stock  cultures.  The  pots  containing 
the  plants  were  placed  in  the  ice  box  on  a wet  earthen  plate  over 
which  a bell  jar  was  inverted.  The  plants  were  usually  left  in  the 
ice  box  from  two  to  twelve  hours,  so  that  ample  time  might  be 
provided  for  the  conidia  to  germinate.  December  6,  two  pots, 
A.  and  B.,  were  treated  as  outlined  above  and  became  heavily  in- 
fected by  December  17.  On  this  date  the  pots  were  placed  on  an 
isolated  bench  where  plenty  of  sunlight  was  available  for  normal 
development  of  the  plants,  to  learn  whether  or  not  the  fungus 
would  maintain  itself  under  green  house  conditions  when  the 
plants  were  closely  crowded  together.  On  December  28  about  half 
of  the  plants  in  the  pots  had  died.  These  were  carefully  pulled 
out  and  more  seed  sown  in  these  same  pots  among  the  living 
plants.  In  a short  time  a new  crop  of  plants  was  at  hand  in  the 
place  where  the  dead  plants  had  been.  A supply  of  young  host 
plants  for  the  fungus  was  thus  provided.  On  this  same  date,  a 
third  pot,  C.,  thirteen  days  old  and  not  infected,  but  otherwise 
exactly  like  the  two  described,  was  placed  as  close  to  the  other 
two  as  possible. 

Observations  were  made  on  January  8 and  it  was  found  that 
five  small  pustules  had  appeared  on  the  plants  in  pot  C.  On 
January  11,  no  changes  were  observed  in  the  extent  of  infection. 
Three  pots,  D.,  E.,  and  F.,  of  mixed  radishes  eight  days  old  and 
not  infected  were  put  with  the  above  three,  making  six  in  all. 
The  next  day  two  six  inch  pots  of  mixed  radishes,  G.  and  TI.,  nine 
days  old,  were  placed  with  the  rest,  all  in  a group  around  the 
infected  plants.  Observations  on  January  21  showed  that  pot  C. 
had  about  10  per  cent  of  its  leaves  infected.  Four  more  pots,  I., 
J.,  K.,  and  L.,  containing  young  seedlings  eighteen  days  old, 
thickly  sown  in  large  pots,  were  added  to  the  group.  January  24 
two  of  the  pots,  eight  days  old,  D.,  and  E.,  showed  two  pustules 
each.  The  following  day  the  third,  F.,  showed  one  pustule.  On 
the  same  day  the  pots  nine  days  old,  G.  and  H.,  showed  infection, 
each  pot  having  four  pustules. 

On  February  5,  two  pustules  had  appeared  on  each  of  the  two 
pots,  I.  and  J.,  placed  on  the  bench  on  January  21.  On  the  fol- 
lowing day  the  third  one,  K.,  showed  five  pustules,  and  the  fourth, 
L.,  nine  pustules.  Daily  observations  were  made  of  the  pots  of 
plants  as  they  were  watered.  The  disease  spread  gradually,  the 


Experiments  on  Spore  Germination.  49 

number  of  infected  areas  increasng  as  well  as  the  number  of 
pustules  in  each  area.  As  the  cotyledons  died  the  young  leaves 
became  infected.  On  February  26,  it  was  evident  that  the  dis- 
ease had  reached  its  maximum.  It  was  estimated  that  15  per  cent 
of  the  leaves  were  infected.  The  spread  of  the  fungus,  however, 
had  hardly  kept  pace  with  the  growth  of  the  plants.  The  leaves 
increased  in  number  faster  than  the  fungus  spread. 

The  disease  gradually  decreased  from  this  time  on.  On  March 
20,  it  was  found  that  about  5 per  cent  of  the  leaves  were  infected. 
The  plants  now  were  six  inches  high  with  six  to  ten  leaves  each 
and  the  number  of  pustules  had  decreased  considerably.  At 
least  40  per  cent  of  the  plants  in  the  six  inch  pots  were  pulled  out 
and  seed  sown  among  the  remaining  plants. 

Observations  were  made  from  time  to  time  on  the  twelve  pots 
of  plants  and  the  fungus  was  found  to  be  gradually  decreasing 
in  amount.  On  April  8,  a damping  off  fungus  attacked  the  young 
plants  just  at  the  surface  of  the  soil,  causing  them  to  fall  over  and 
dry  up.  The  young  crop  had  become  infected  in  every  pot,  but 
because  of  the  damping  off  fungus,  the  infection  was  not  as  ex- 
tensive as  had  been  the  case  before.  Three  pots  were  so  badly 
injured  that  they  were  discontinued.  The  large  leaves  of  the 
plants  in  the  pots  were  shading  the  surface  of  the  soil  too  much 
for  healthy  growth  of  the  young  plants.  To  relieve  this  condi- 
tion, the  tops  of  all  the  plants  in  the  pots  were  cut  down  even 
with  those  of  the  young  plants.  It  is  quite  evident  that  this  is  a 
method  that  can  be  used  to  maintain  the  white  rust  under  green 
house  conditions.  Two  infected  pots  of  crowded  seedlings  in- 
fected ten  more  standing  near  them.  At  no  time  was  more  than 
15  per  cent  of  the  leaves  infected  and  as  the  plants  grew  older, 
the  amount  of  white  rust  decreased.  The  inoculation  period  in 
these  experiments  varied  from  eleven  to  sixteen  days,  due  pos- 
sibly to  lack  of  optimum  conditions  for  germination.  This  was 
longer  than  was  required  when  the  stock  cultures  were  chilled. 

An  attempt  was  made  to  show  whether  or  not  the  white  rust 
could  be  maintained  directly  cn  the  green  house  benches  without 
chilling  the  seedlings  in  the  beginning  as  was  done  in  the  above 
experiment.  The  radish  seed  was  sown  directly  on  the  bench  and 
not  in  pots.  It  was  thought  that  possibly  the  disease  might  do 
better  under  these  conditions.  On  November  1,  four  varieties  of 
radish  were  sown  in  a box  built  on  the  bench,  which  was  8 inches 
wide  and  3 feet  long.  When  the  plants  were  nine  days  old  and 


50 


Wisconsin  Experiment. Station. 


showed  twm  cotyledons  they  were  sprayed  with  water  and  inocu- 
lated with  Cystopus  candidus  spores  from  “ Early  scarlet  globe” 
radish.  On  November  18,  one  leaf  was  found  having  one  pus- 
tule. November  24,  eleven  cotyledons  showed  infection.  From 
that  time  on  the  disease  increased  gradually.  On  December  16, 
it  was  estimated  that  about  300  of  the  2000  plants  showed 
white  pustules. 

December  29,  the  pots  of  plants  that  had  been  inoculated  but 
not  chilled  and  the  plants  in  the  box  on  the  bench  showed  about 
the  same  degree  of  infection. 

A record  of  the  temperature  was  kept  by  placing  a self  regis- 
tering thermometer  as  near  the  box  of  plants  as  possible.  The 
range  of  temperature  was  about  12°  C.  for  the  first  thirteen  days. 
During  the  day  the  temperature  was  about  27°  C.  and  at  night 
about  15°  C.,  showing  that  temperature  conditions  are  some- 
times favorable  in  the  greenhouse. 

The  plants  in  this  experiment  were  given  the  most  favorable 
conditions  possible  and  they  showed  normal  growth.  This  was 
evident  from  the  dark  green  color  on  the  cotyledons  and  leaves. 
When  the  radish  plants  are  sickly  and  not  doing  well  in  the  green 
house  the  infected  cotyledons  and  the  leaves  tend  to  become  varie- 
gated and  fall  off.  None  of  these  symptoms  were  noted  in  the 
plants  studied  in  this  experiment.  The  experiment  indicates 
that  it  is  impossible  to  get  any  such  development  of  the  disease 
as  was  secured  by  chilling  the  plants  at  the  time  of  inoculation. 
Still  the  disease  appeared  without  chilling  and  increased  in 
amount  until  about  300  of  the  plants  became  infected.  There- 
fore, it  is  possible  to  maintain  the  white  rust  under  green  house 
conditions  by  the  method  suggested  in  this  experiment. 


Experiments  on  Spore  Germination. 


51 


Table  VIL — Record  op  Maintaining  Ten  Infected  Stock  Cultures. 


j «_j  1 Name  j 

Cult. 

Variety  of  radish. 

A ere 
Cult. 

Tyne 
in  ice 
box. 

| Date 
inoc. 

Date 

inf. 

Date  re- 
planted. 

Results. 

Ne  Plus  Ultra 

days 

5 

18 

42 

46 

7-7 

S3 

118 

148 

164 

192 

Hrs. 

m 

6 

6 

19 

12 

Sept.  29 
Oct.  12 
Nov.  5 

Dec.  17 
Feb.  23 

Oct.  6 
Nov.  9 
Jan.  24 

Apr.  8 

Oct.  12 

Dec.  11 
Jan.  13 
Mch.  11 

Heavy  inf. 

Young-  leaves  well 
infected. 

Well  infected. 

Second  crop  heavily 
infected. 

Well  infected. 

Heavy  inf. 

Old  plants  with  little 
inf.;  younger  plants, 
plenty  of  inf. 

Old  pi.  about  to  bloss- 
om. Tending  to 

shade  young  pi.  Not 
well  inf. 

Two  old  pi.  in  blos- 
som. Pot  filled  with 
soil. 

K. 

Crimson  Giant 

9 

6 

Dec.  25 

Nov.  9 

Well  infected. 

43 

Dec.  7 

Well  infected. 

53 

19 

Dec.  17 

Dec.  31 

Young  heavily  inf. 

80 

Jan.  13 

Well  infected. 

121 

1 12 

Feb.  23 

Low  in  inf. 

Mch,  11 

Young  crop  well  inf. 

Mch.  24 

6 big  plants  about  to 

blossom,  other  pi. 

shaded.  Poor  inf. 

165 

Apr.  8 

One  pi.  in  flower. 

Others  in  bud.  Poor 

inf. 

L. 

New  White  Chinese 

6 

6 

Dec.  17 

Dec.  29 

Well  infected. 

27 

20 

Jan.  13 

Jan.  13 

Jan,  13 

Disease  increased  in 

amount. 

41 

12 

Feb.  23 

Feb.  23 

Well  infected. 

Ne  Plus  Ultra.1. 

57 

Mch.  11 

Young  crop  well  inf. 

M. 

Scarlet  turnip,  white  tip. 

5 

6 

Dec.  17 

Jan.  2 

Nov.  1 

Well  infected. 

Ne  Plus  Ultra 

Jan.  13 

Coty  dead.  Young  pi. 

becoming  inf. 

12 

Feb.  23 

Mch.  11 

Young  pi.  well  inf. 

Infection  decreased. 

Ne  Plus  Ultra 

86 

Young  plant  inf.,  old 

also  tho  not  as  much 

as  young. 

N. 

Old’s  Snowball 

9 

8.5 

Dec.  21 

Jan.  2 

Well  infected. 

Jan.  13 

Well  infected. 

83 

12 

Feb.  23 

Well  inf.  Pot  broken. 

0 . 

Cincinnati  Market 

i 

23 

Dec.  13 

Dec.  26 

Well  infected. 

NePlus  Ultra 

95 

Mch.  11 

Pi,  5 in.  high.  5%  of 

pi.  inf. 

P. 

Mixed  radish 

8 

7 

Jan.  11 

Jan.  22 

Well  infected. 

22 

Feb.  23 

Feb.  23 

Well  infected. 

Ne  Plus  Ultra 

67 

22 

Mch. 

Infection  low. 

Name 
I Cult. 


52 


Wisconsin  Experiment  Station. 


Table  VII  Continued. — Maintaining  Ten  Infected  Stock  Cultures 


Variety  of  Radish. 


Old's  Twenty  Day 


Age 

Cult. 

Time 
in  ice 
box. 

Date 

inoc. 

Date 

inf. 

Date  re- 
planted. 

7 

24 

Dec.  13 

Dec,  23 

.Tan.  22 

• 9 

Feb.  25 

Jan.  13 

95 

22 

Mch.  11 

Results. 


Well  infected. 

Less  than  A of  pi.  inf. 
Young  crop  well  inf. 


E. 

Winter 

China  Rose 

11 

78 

1 

24 

22 

Jan.  3 
Feb.  23 
Mch.  11 

.Jan.  12 

j . , 

Feb.  25 

Well  infected. 

S. 

Mixed  Radish  . . 

9 

24 

Jan.  12 

Jam  20 

Well  infected. 

24 

Feb.  23 

75%  of  leaxes  inf. 

Feb.  25 

75%  of  leaves  inf. 

_ 

67 

7 

Mch.  11 

Decreased  in  inf, 

Summary  of  Table  VII 

On  the  basis  of  the  experiment  with  stock  cultures  just  de- 
scribed the  following  method  for  maintaining  stock  cultures  was 
marked  out.  Good  infections  were  secured  by  spraying  the 
spores  on  the  young  seedlings  in  a crowded  six  inch  pot  with  an 
atomizer  and  then  setting  the  pot  on  a wet  earthen  plate  over 
which  a bell  jar  was  inverted.  Under  these  conditions  the  pots 
of  plants  were  placed  in  the  ice  box  where  the  temperature  was 
about  10°  C.,  long  enough  to  allow  the  conidia  to  germinate. 
After  this  they  were  placed  on  a bench  in  the  green  house.  As 
the  fungus  continued  to  kill  the  young  seedlings,  others  were 
supplied  by  sowing  more  seed  among  the  remaining  plants. 
When  this  new  crop  came  up,  the. pots  were  again  placed  in  the 
ice  box  as  described  above  so  that  the  young  plants  might  become 
infected.  It  will  be  convenient  in  the  following  work  to  speak 
of  a six  inch  pot  thickly  sown  with  radish  plants  infected  with 
Cystopus  candidus,  as  a stock  culture.  Nine  of  these  stock  cul- 
tures were  maintained  in  this  experiment,  each  designated  by  a 
capital  letter  from  J to  S.  In  all  the  stock  cultures  from  J to  S 
good  infections  were  maintained  by  chilling  and  reseeding  when- 
ever the  disease  showed  signs  of  disappearing.  These  stock  cul- 
tures became  the  source  for  the  conidia  used  in  the  subsequent 
experiments  up  to  the  time  when  the  plants  were  about  to  bloom. 
In  stock  cultures  J and  K good  infections  were  kept  in  the  same 
pot  for  192  and  165  days  respectively.  On  March  8,  as  a result 
of  the  pots  becoming  so  filled  with  soil  from  continued  reseeding, 
it  was  impossible  to  grow  more  new  plants  among  the  old  ones. 


Experiments  on  Spore  Germination. 


53 


At  this  time  the  old  plants  in  the  stock  cultures  were  in  blossom 
and  the  leaves  were  large  and  broad.  This  condition  was  not  con- 
ducive to  the  growth  of  young  plants  in  the  same  pot.  Even  in 
this  stage  of  development,  the  plants  were  not  free  from  disease 
although  the  pustules  were  much  less  abundant  than  when  the 
plants  were  younger.  The  stock  cultures  from  L to  S were  all 
younger  than  J and  K,  and  on  March  11,  all  the  plants  were 
about  to  blossom.  The  white  rust  was  evident  in  every  stock 
culture  but  was  not  as  plentiful  as  when  the  plants  were  smaller. 
Had  it  not  been  that  it  was  desired  to  make  a study  of  the  pro* 
duction  of  oospores,  the  larger  plants  might  have  been  cut  off, 
and  the  culture  inoculated  again  whenever  the  disease  showed 
signs  of  marked  decrease  in  amount. 

Stock  culture  J was  chilled  in  the  ice  box  five  times,  reseeded 
four  times  and  maintained  a good  infection  for  192  days  or  until 
the  plants  blossomed. 

Stock  culture  K was  chilled  in  the  ice  box  three  times  and  re- 
seeded three  times  during  the  165  days.  Infected  plants  were 
always  abundant  until,  the  old  plants  blossomed. 

The  remaining  stock  cultures  L to  S were  not  as  old,  but  in 
every  case  they  were  chilled  at  least  twice  and  reseeded  from  one 
to  three  times.  Here  also,  well  infected  stock  cultures  were  main- 
tained until  March  11,  when  the  plants  grew  tall  and  began  to 
blossom.  This  is  undoubtedly  a method  by  which  white  rust 
can  be  maintained  abundantly  under  green  house  conditions 
with  the  use  of  only  a few  pots.  The  full  data  as  to  these  cul- 
tures are  given  in  Table  V. 

To  fully  test  the  value  of  chilling  in  maintaining  stock  cultures 
of  Cystopus,  parallel  cultures,  chilled  and  not  chilled,  were  made 
during  a period  of  1 16  days.  Chilling  at  the  start  a crowded  stock 
culture  of  Raphanus  sativus  such  as  is  described  above  and  inocu- 
lated with  Cystopus  candidus  from  the  radish  undoubtedly  causes 
a larger  per  cent  of  infection  than  when  the  stock  cultures  are 
not  chilled.  Six  inch  pots  were  thickly  seeded  with  radish  so 
that  each  pot  contained  about  two  hundred  seedlings  when  it 
was  about  ten  days  old.  The  plates  except  VII  and  VIII  are 
photographs  of  such  cultures.  At  this  time  the  stock  cultures 
consisting  of  two  hundred  seedlings,  were  inoculated  with  con- 
idia  of  Cystopus  candidus  from  Raphanus  sativus.  The  conidia 
were  sprayed  on  the  young  seedlings  with  an  atomizer.  Fol- 
lowing this  treatment,  the  stock  cultures  were  placed  on  a wet 


Table  ^ III.  Effect  of  Chilling  Stock  Cultures  of  Ruphanus  sativus  Inoculated  with  Conidia  from  Cystopus  candidus 


54 


Wisconsin  Experiment  Station. 


Control  not  chilled. 

Date 

photo- 

graphed. 

Nov.  17. 
Jan.  5. 
Jan.  18. 
Jan.  18. 
Jan.  25. 
Nov.  29. 
Nov.  29. 
Dec.  19. 
Dec.  27. 
Jan.  25. 
Dec.  27. 
Jan.  25. 
Dec.  29. 
Dec.  27. 

. 1 Ext.  inf 

Date  inf 

Nov.  15. . 
Dec.  31... 

Jan.  12. . 
Jan.  21. . 
Nov.  18.. 
Nov  .18.. 
Dec.  12.. 
Dec.  19.. 
Jan.  22.. 
Dec.  26.. 
Jan.  21.. 
Dec.  12. . 
Dec.  19.. 
Mar.  1... 
Mar.  8 . . . 
Mar.  8.,. 
Mar.  5... 
Jan.  22... 
Jan.  22... 
Jan.  24... 
Jan.  10  . . 
Jan.  7 

Date 

photo- 

graphed. 

. Nov.  17. 
Jan.  5.. 
Jan.  18. 
Jan.  18. 
Jan.  25. 
Nov.  17. 
Nov.  17. 
Dec.  19.. 
Dec.  27.. 
Jan.  25. 
Dec.  27.. 
Jan.  25. 
Dec.  29.. 
Dec.  27.. 

1 



Ext.  inf. 

mii444444444444444444 

Date  inf. 

Nov.  5 

Dec.  31... 
Jan.  12... 
Jan.  12... 
Jan.  24... 
Nov.  15.. 
Nov.  15.. 
Dec.  12... 
Dec.  17... 
Jan.  22... 
Dec.  23... 
Jan.  24... 
Dec.  12... 
Dec.  17... 
Mar.  3. . . 
Mar.  1... 
Mar.  3... 
M ar.  1 ... 
Jan.  18... 
Jan.  18... 
Jan.  19... 
Jan.  7 .... 
Jan.  7.... 

Period  of 
refrigeration . 

a 

S 

0) 

Eh 

2^222  ■’  ;®>v50©rc<3im®oooooo.— '—1 

Hours. 

« ^ — S8g 

Source  of  conidia. 

Ne  Plus  Ultra 

Ne  Plus  Ultra 

Ne  Plus  Ultra 

Ne  Plus  Ultra 

Ne  Plus  Ultra 

Ne  Plus  Ultra 

Ne  Plus  Ultra 

Ne  Plus  Ultra 

Ne  Plus  Ultra 

Ne  Plus  Ultra 

Ne  Plus  Ultra 

Ne  Plus  Ultra 

Ne  Plus  Ultra 

Ne  Plus  Ultra 

Ne  Plus  Ultra 

Ne  Plus  Ultra 

Ne  Plus  Ultra 

Ne  Plus  Ultra 

Ne  Plus  Ultra 

Ne  Plus  Ultra 

Ne  Plus  Ultra 

Ne  Plus  Ultra 

Ne  Plus  Ultra 

Variety  of  radish. 

' 

White  Icycle 

1 Triumph 

Triumph 

China  Rose  Winter 

Mixed 

NePlus  Ultra 

NePlus  Ultra 

NePlus  Ultra 

| NePlus  Ultra 

Olds’  Golden  Globe 

Olds’  Twenty  Day 

Mixed 

NePlus  Ultra 

NePlus  Ultra 

NePlus  Ultra 

NePlus  Ultra 

NePlus  Ultra 

NePlus  Ultra 

Mixed 

Mixed 

First  and  Best 

NePlus  Ultra 

NePlus  Ultra 

Date. 

Oct.  29... 
Dec.  21... 

Jan.  3 

Jan.  3 

Jan.  11.. . 
Nov.  8... 
Nov.  8..., 
Dec.  3..., 

Dec.  8 

Jan.  10... . 
Dec.  13.... 
Jan.  11.... 

Dec.  3 

Dec.  8 

Feb.  22... 
Feb.  22... 
Feb.  22... 
Feb.  22... 
Jan.  12.. . . 

Jan.  12 

Jan.  12., . . 
Dec.  28.... 
Dec.  28... . 

No.  of 
exp. 

Experiments  on  Spore  Germination. 


55 


earthen  plate  under  a bell  jar  and  placed  in  the  ice  box  for  a 
period  varying  from  six  to  twentyfour  hours.  When  the  stock 
cultures  were  removed  from  the  ice  box  they  were  placed  on  a 
bench  in  the  green  house  and  in  from  seven  to  twelve  days  the 
fungus  made  its  appearance.  The  controls  used  in  each  experi- 
ment were  treated  in  the  same  manner  as  the  stock  cultures  except 
that  they  were  not  chilled  but  placed  directly  on  a bench  in  the 
green  house.  The  results  were  photographed  when  the  pustules 
were  fully  developed  and  about  to  burst. 

The  extent  of  infection  secured  by  the  chilling  method  can  best 
be  seen  by  referring  to  the  plates.  Plates  I and  II  show  two 
stock  cultures  of  radish  seedlings  that  were  inoculated  when  the 
seedlings  were  ten  days  old.  In  seven  days,  the  cultures  began 
to  show  infection.  Four  days  later,  or  eleven  days  after  inocu- 
lation, the  conidial  pustules  were  fully  developed  and  the  results 
photographed  to  show  the  difference  in  the  extent  of  infection 
between  cultures  chilled  and  not  chilled.  Pustules  developed  on 
both  the  upper  and  lower  side  of  the  cotyledons  but  only  on  the 
lower  side  of  the  first  true  leaves,  which  can  be  seen  in  the 
plates.  November  8,  two  more  stock  cultures  were  inoculated 
and  became  infected  November  15.  The  results  were  photo- 
graphed November  17,  two  days  later  (Plates  III  and  IV). 
Here,  'again,  it  will  be  noted  that  chilling  produces  the  more 
abundant  infection.  These  two  stock  cultures  were  kept  as  were 
many  others  as  a source  of  conidia  for  future  inoculation. 
November  29,  or  twentyone  days  after  inoculation,  the  stock 
cultures  shown  in  Plates  III  and  IV  were  photographed  again  to 
show  the  further  development  of  the  fungus  as  well  as  the  effects 
on  the  host  plants  (Plates  V and  VI).  The  radish  seedlings  in 
the  chilled  culture  were  being  killed  rapidly  by-  the  fungus  while 
in  the  control  (Plate  VI)  the  plants  are  healthy  with  no  marked 
increase  in  the  amount  of  white  pustules.  It  was  very  evident 
in  this  experiment  as  wrell  as  in  all  the  others  that  more  abundant 
infection  had  occurred  in  the  cultures  that  were  chilled.  The 
same  striking  results  were  secured  when  the  radish  seedlings 
were  grown  in  small  pots  (Plate  VII).  In  these  experiments, 
radish  seedlings  were  grown  in  three-inch  pots  and  treated  as 
described  above.  The  effect  of  chilling  can  be  readily  seen. 
The  results  shown  in  Plate  VII  suggest  that  an  extensive  infec- 
tion is  not  dependent  upon  the  crowded  condition  of  seedlings 
in  the  stock  cultures,  but  upon  chilling.  Not  only  do  the  coty- 


56  Wisconsin  Experiment  Station. 

ledons  become  heavily  infected  by  the  chilling'  method,  but  also 
the  leaves.  The  curled  and  hypertrophied  leaves  can  be  seen  in 
Plate  VIII.  The  leaves  are  as  readily  infected  as  the  cotyledons 
of  the  radish.  In  Lepidium  sativum  only  the  cotyledons  can 
become  infected  according  to  DeBary  (1863;  27). 

Twenty-four  tests  with  a control  for  each  trial  were  made,  be- 
tween October  29,  1909,  and  February  22,  1910.  Experiments 
have  been  continued  since  the  last  named  date  with  the  same 
striking  results.  Plates  I and  X show  stock  cultures  made  from 
October  29,  1909,  to  June  6,  1910,  and  photographed  when  the 
fungus  was  well  developed.  In  every  case  in  which  the  stock 
cultures  were  chilled,  a heavy  infection  resulted;  while  the  con- 
trols, except  in  one  case,  showed  but  little  development  of  the 
white  rust.  The  control  in  experiment  No.  4 on  January 
11,  showed  an  exceptional  development  of  infection.  It  was  as 
heavily  infected  as  the  stock  culture  that  was  chilled  which  again 
shows  that  the  conditions  favorable  for  infection  do  occasionally 
occur  in  the  green  house,  as  has  already  been  pointed  out.  It 
might  naturally  be  asked  whether  chilling  has  the  same  effect 
in  the  spring  and  summer  as  in  the  fall  and  winter.  Experi- 
ments have  been  carried  on  continuously  since  February  22 
(although  the  results  are  not  tabulated)  until  June  6,,  1910, 
with  the  same  marked  results  as  were  obtained  during  the  fall 
and  winter  of  1909.  The  above  data  lead  me  to  conclude  that 
chilling  strongly  favors  the  infection  of  radish  plants  with  Cys- 
topus  candidus. 


THE  RELATIVE  SUSCEPTIBILITY  OF  COTYLEDONS 
AND  LEAVES  TO  CYSTOPUS  CANDIDUS 

In  the  preceding  experiments  it  was  quite  impossible  to  deter- 
mine whether  any  marked  degree  of  difference  of  susceptibility 
existed  between  cotyledons  and  leaves  of  the  radish.  DeBary 
found  that  it  was  only  the  cotyledons  that  showed  any  marked 
degree  of  susceptibility.  In  order  to  test  this  point  for  the  coty- 
ledons and  leaves  of  radish,  shepherd’s  purse,  white  mustard, 
and  garden  cress,  the  following  experiments  were  planned. 

All  the  plants  utilized  in  this  series  of  experiments  were  grown 
in  the  green  house  and  were  in  all  cases  free  from  infection  at 
the  putset.  Twelve  three-inch  pot§  were  seeded  with  radish  on 


Experiments  on  Spore  Germination. 


57 


October  15,  each  pot  containing  only  two  plants.  On  November 
11  the  plants  had  lost  their  cotyledons  and  the  first  leaves  were 
two  to  three  inches  long.  At  this  time  the  twentyfour  plants 
were  inoculated  and  chilled  and  on  November  23,  twelve  of  the 
plants  showed  infection.  On  the  following  day  twenty  of  the 
twentyfour  plants  inoculated  were  infected.  There  was  not  the 
slightest  evidence  that  the  infection  was  abnormal  as  the  pustules 
were  abundant  on  each  leaf  and  the  leaves  were  becoming  badly 
distorted.  On  September  15,  five  plants  of  Capsella  that  had 
grown  from  seed  collected  outdoors  and  planted  in  the  green 
house  were  in  blossom.  At  this  time  the  plants  were  inoculated 
with  conidia  from  Capsella  and  September  26,  three  of  the 
plants  were  infected  on  both  stem  and  leaves.  On  the  following 
day  the  leaves  and  young  fruits  of  the  two  remaining  plants  also 
became  infected.  A stock  culture  of  at  least  fifty  white  mustard 
plants  that  were  planted  September  1 in  the  green  house  were 
inoculated  September  23  when  the  plants  were  six  inches  tall. 
They  were  healthy  and  the  cotyledons  had  fallen  off.  On  the 
last  named  date,  the  culture  was  inoculated  with  spores  from 
the  white  mustard  and  chilled.  On  October  12,  at  least  75  per 
cent  of  the  Laves  on  the  plants  were  infected. 

I have  never  collected  Cystopus  on  garden  cress  so  it  was  im- 
possible to  test  this  host  with  the  Cystopus  growing  on  it  out- 
doors ; but  I have  inoculated  garden  cress  with  spores  from  Cap- 
sella. Three  three-inch  pots  of  garden  cress  containing  eighteen 
plants  that  were  four  inches  high  and  without  cotyledons  were 
inoculated  October  15.  Infection  resulted  October  27.  Twelve 
of  the  plants  became  infected.  From  the  above  results  it  is 
clear  that  the  leaves  as  well  as  the  cotyledons  of  radish,  shep- 
herd’s purse,  white  mustard,  and  garden  cress  are  readily  in- 
fected  with  Cystopus  candidus. 


SUSCEPTIBILITY  OF  DIFFERENT  VARIETIES  AND 
SPECIES  OF  RAPHANUS  TO  CYSTOPUS  CANDIDUS 

One  object  in  developing  a method  of  germinating  the  conidia 
of  Cystopus  with  certainty  and  in  abundance  was  to  provide 
means  for  attacking  the  question  as  to  the  existence  of  so-called 
physiological  species  in  the  genus.  It  seemed  desirable  also  to 
test  the  relatiye  susceptibility  of  different  varieties  qf  radish 


58 


Wisconsin  Experiment  Station. 


to  Cystopus.  For  this  latter  purpose  twentytwo  varieties  of 
radish  were  grown  and  inoculated.  In  these  experiments  on 
different  varieties  of  radish,  three-inch  pots  were  used  and  about 
ten  seeds  of  the  same  variety  were  planted  in  each  of  the  three 
pots.  When  the  plants  were  eight  to  twelve  days  old  all  of  the 
plants  in  each  pot  except  three  of  the  healthiest  were  pulled  out. 
At  this  time  each  plant  was  about  1%  inches  high  and  had  two 
well  developed  cotyledons  but  no  true  leaves.  These  plants 
were  then  inoculated.  In  testing  each  variety,  at  least  nine 
plants  were  inoculated.  In  Table  IX  are  given  the  results  ob- 
tained from  inoculating  twentytwo  varieties  of  radish  with 
conidia  taken  from  the  varieties  Ne  Plus  Ultra,  White  Icicle,  or 
Crimson  Giant.  In  every  experiment  except  those  started 
November  9 and  November  12,  Ne  Plus  Ultra  was  used  as  the 
source  of  conidia.  In  all,  ninetyseven  inoculations  were  made 
and  in  ninetyfive  cases,  infection  appeared.  Controls  of  Ne  Plus 
Ultra  of  the  same  age  and  under  the  same  conditions  were  kept 
in  every  experiment  and  always  showed  abundant  infection. 
97%  per  cent  of  the  cotyledons  inoculated  became  infected. 
This  would  suggest  very  little  immunity  for  any  of  the  twenty- 
two  varieties  of  radish  tested,  to  Cystopus  Candidas.  It  is 
quite  evident  that  the  same  form  of  Cystopus  Candidas  can  grow 
on  all  the  varieties  of  radish. 

Tests  were  next  made  to  determine  whether  or  not  a different 
species  of  Raphanus  (Raphanus  caudatus)  could  be  infected  with 
conidia  of  Cystopus  from  Raphanus  sativus.  Nine  plants  grow- 
ing in  three  different  pots  were  inoculated  and  all  became  in- 
fected showing  that  Cystopus  Candidas  on  Raphanus  sativus  is 
not  limited  to  this  species,  but  can  also  infect  Raphanus  cauda- 
tus. Many  tests  have  been  made  with  the  above  species  since 
this  experiment  with  similar  results. 


Table  IX. — Relative  Susceptibility  of  Different  Varieties  of  Baphanus  sativus  to  Cystopus  candidus  from:  Baphanus 

sativus,  Variety  Ne  Plus  Ultra  and  Others. 


Experiments  on  Spore  Germination. 


59 


O o 


acsaflgflSEJcscflcccc 


Dead. 

OrHOOOOOOO'MOrHr^O'^OOrH^rH(MOOOTH(MOO(MOOTHOOOOOOO 

xn 

H 

3 

d 

Not  1 
inf. 

HOHHOlMO^MHNOHWNOlMCNHON^OOWOJ^^OHHOHfOWnHO 

q 

CO 

H 

P3 

Inf. 

Date. 

in«M/!i(5U5inHOO)OOOOOWaiffittNOwHWW^ffl!OBNHOHOHHOHOifi 

r-  rH  (M  NWNWNTHrtrtr^HWWMNNMWWMN  CO  CO  CO  CO 

> > > > > > > > > > > >>>>>>>>  > t>"  > > > > > >■'  >'  > o>  6 > 6 © 1>  d > o' 

00000000000000000000000000000©0©0®©0®0© 

•OJ'-O-O'-O- 

■ oo  to  to  iiO  t 

• 03  CO  CO  Lft  CO  CO  CO  < 


NNNNNNNIMN1 


' — 1 ’ — 1 ' — 1 ' — 1 ^ ■ — 1 ’ — 1 t — ? — l>-  t-  I-  Cl 


7>  I 


°pS 


lO  K5  UO  KO  IfJ  lf3  1ft  ift  UO  K5iOir5ifiiOiOiOK5ifl«5ii5iflinK5 

(■—l  WMWKNOl  WW  cj 


>i  o © cococococococOKOiouoiouoiomioioiotfouoifOioioicsioioxoiouoioiciiOinioioioiOKOiom 


O-fa  3^ 

. 5 o © 
o2  2^ 
a.S~ 


C5CiC53;OJOiG5<MCOCOCOCOrOCD:00505CTacOC5CT5Ci<MCiS'lCi0505C:!COCOCCCOCOCOCDeOCOOi 


O3c3o3o3c3c3c3c3o3 


pO0)0)d. 


©^2 . 
V Of 


O © O _ 

C/3t/)r/3C/5V3CC72t/5»?a2MC/3(/3C/3!Zll~(HHI-Ha7;tnc/)V;f 

2222222222=2222®  ©©32222  ®©aa;^.a.a^a.a 

PhPhPm P-da-,p^p_iPHpuPLiPLiP_tP-«G^pMi3^i5  SOmP-iP-iPM^^P^PLiQ-iP-iPhP-iPhC^PhP-i 

©©©©©©©©©©©©©©©•^af'^'E®©®®-'- 


© 

+j 

2 

^ ".S— 

«h  xnp'  ci 

a ©0x2 


,fa  ®T! 

T2  43  IH 


2 3 ^©g-gS 
o g.2  n £ M 

rt  a?  c3  'fa  bo  q &b 

2 s = SoO  §-g  § $ 8i5rD  5^  b 


- c3 

'.Mi 
• S3 
: ® 


S :fa 


T3^h'— i . -hV 

bt-^  © 

> ci  d Q c3  5> 


O O --H  gj  © © ' 


||g| 


© >> 

■ 


a ; a 

’3  :a 


s v s 
Sfe-Sfe-S 


7^  fejs 


o ^CPiSS^I^gbo^  o §2  *s* 

■ — 1 GMix!  ©_,  © «s  s qs  © 2 a 


q’g  5 ©tj.21, 


^CO^CKm  £.0  £ 

_ „ „ .fa..  » q 

fei£i£i2  bu  m ,53  q e <-,  « 

••=  §2  b.5  © ©£  g-c  ©S22-C2S-C  § oS 


« © © 2 ©.a  Krt  o.fa  V.fa 
O.^  o ~2|  g | © | O 

~eh2hm 


t . c3  © ^ t>,  © © 

2 ^'Vjq'qx: 


aQfcfc0£W00bfc&5tf<nttfc000«00HJiJ0£0£H0^0E 


q • 

H :e 

43  : , 

© . 

>i  c3  * 

S3  O . 
r&m  .c_^, 

.!  43  to  43  m q 

« 03  © 03  © <S 

c®cq2ffl  o 

©'r_-TH_  m 

ssls'gg 

ehhSbSS 

,52  J£  rrt  "» 
22  fa2  U CS 
OOfeOfaM 


43  43- 43*  43’  ©>'  4J  4J-  > > >>>•>>■>>>  > > > >•  > t>  >>>>>>>■>>>>>  >.  > 

©©0©00o0000000000000000000000000000000 


{OWMMmMM^CJNHHNNNMeOCCWWMM'tM- 


IHHHNWi 


60 


Wisconsin  Experiment  Station. 


SUSCEPTIBILITY  OF  OTHER  CRUCIFERS  TO  CYSTO- 
PUS CANDIDUS  FROM  THE  RADISH 


Tests  were  made  to  learn  if  the  spores  of  Cyst  opus  candidus 
from  the  radish  can  infect  turnips  (Brassica  rapa).  The  tur- 
nip plants  used  in  this  series  of  experiments  were  grown  in  the 
same  way  as  the  radish  plants,  as  explained  in  connection  with 
Table  VII.  Three  healthy  plants  were  allowed  to  grow  in  each 
pot  and  these  were  inoculated  by  spraying  them  with  the  spores 
of  Cyst  opus  candidus  from  the  radish.  The  spores  were  sprayed 
on  with  an  atomizer  and  the  plants  chilled  in  the  usual  manner. 

As  shown  in  Table  X,  ten  varieties  of  turnips  (Brassica  rapa ) 
were  inoculated  with  conidia  of  Cystopus  candidus  from  the 
radish,  variety  Ne  Plus  Ultra.  In  each  variety,  at  least  nine 
plants  were  inoculated,  while  in  the  case  of  Snowball,  eighteen 
plants  were  tested.  This  would  mean  that  eighteen  or  more 
cotyledons  were  inoculated.  These  experiments  extended  from 
November  13  to  January  28,  and  at  no  time  did  any  infection 
result,  although  in  every  trial  the  controls  were  infected.  It 
was  impossible  to  infect  any  of  the  ten  varieties  of  turnips  with 
Cystopus  candidus  from  Raphanus  sativus , variety  Ne  Plus 
Ultra.  These  results  suggest  that  there  may  be  a physiological 
species  of  Cystopus  candidus  occurring  on  radish  and  turnip 
respectively. 


Table  X. — Relative  Susceptibility  of  Different  Varieties  Brassica  rapa 
to  Cystopus  candidus  from  Raphanus  sativus , Variety  Ne  Plus  Ultra. 


No.  of  cult,  tested. 

Date. 

Host  Plant. 

Source  of 
conidia. 

I No.  pi.  inoc. 

, No.  controls  inoc. 

Period 
refrig- 
era- 
tion . 

Results 

Date. 

Inf.  coty.  1 1 

Controls.  || 

i 

3 

O 

1 

© 

3 

Nov. 

30. 

Yellow  aberdeen 

Ne  Plus  TTltra. 

9 

5 

4 

16 

Dec.  17,. 

0 

Tnf . 

3 

Nov. 

30. 

Olds’  heavy  copper 

Ne  Plus  Ultra. 

9 

5 

4 

16 

Dec.  17.. 

0 

Inf. 

3 

Dec. 

4.. 

Golden  ball 

Ne  Plus  TTltra. 

9 

5 

5 

17 

Dec.  22.. 

0 

Tnf. 

3 

Dec. 

6.. 

White  flat  dutch 

Ne  Plus  TTltra. 

9 

5 

8 

11 

Dec.  17.. 

0 

Inf. 

3 

Dec. 

28. 

Early  white  milan 

Ne  Plus  Ultra. 

9 

5 

17 

9 

Jan.  13.. 

0 

Inf. 

3 

Dec. 

28. 

Cow  horn 

Ne  Plus  TTltra. 

9 

5 

17 

9 

Jan.  13.. 

0 

Inf. 

3 

Dec. 

28. 

Extra  early  purple  top  milan. 

Ne  Plus  TTltra. 

9 

5 

17 

9 

Jan.  13.. 

0 

Tnf. 

3 

Dec. 

28. 

Purple  top  white  globe 

Ne  Plus  TTltra. 

9 

5 

17 

9 

Jan.  13.. 

0 

Tnf. 

3 

Dec.  28. 

White  egg 

Ne  Plus  TTltra. 

9 

5 

17 

9 

Jan.  13.. 

0 

Inf. 

3 

Jan. 

10. 

Snowball,  

Ne  Plus  TTltra. 

9 

5 

23 

10 

Jan.  22.. 

0 

Tnf. 

3 

Jan. 

22. 

Snowball 

Ne  Plus  TTltra. 

9 

5 

26.5 

12 

Feb.  15.. 

0 

Tnf. 

2 

.T  an. 

28. 

Early  white  milan 

Ne  Plus  TTltra. 

6 

5 

26.5 

12 

Feb.  15.. 

0 

Tnf. 

1 

Jan. 

2S. 

Golden  ball 

Ne  Plus  Ultra. 

3 

5 

26.5 

12 

Feb.  15.. 

0 

Inf. 

No.  cult,  tested. 


Experiments  on  Spore  Germination. 


61 


Summary  of  Table  XI 

An  attempt  was  made  to  infect  three  different  varieties  of 
rutabaga  {Brassica  campestris) , namely:  Olds’  Improved  Purple 
Top,  New  Necklace,  Olds’  Large  White,  with  the  conidia  from 
the  radish,  variety  Ne  Plus  Ultra.  Three  seedlings  were  grown 
in  two-inch  pots  in  the  same  manner  as  described  in  connection 
with  Table  IX.  Controls  were  kept  in  every  experiment  and 
always  became  infected.  Final  observations  were  made  in  the 
case  of  the  different  varieties  of  rutabaga  several  days  after  the 
controls  had  become  infected.  This  was  done  so  as  to  exclude 
any  possibility  of  overlooking  the  disease,  which  possibly  might 
take  longer  to  develop  on  the  rutabaga. 

The  age  of  the  seedlings  inoculated  varied  from  eleven  to 
twentynine  days.  The  plants  had  one  to  five  leaves.  Both  coty- 
ledons and  leaves  were  inoculated.  In  all,  sixteen  separate  trials 
were  made.  In  each  trial,  three  plants  were  inoculated,  making 
a total  of  fortyeight  plants  inoculated,  and  in  no  case  did  infec- 
tion result.  These  experiments  extended  from  September  25, 
1909,  to  January  28,  1910.  Plants  of  different  ages  were  tested. 
It  is  quite  evident  that  Cystopus  candidus  occurring  on  Rapha- 
vus  sativus,  variety  Ne  Plus  Ultra,  will  not  grow  on  Brassica 
campestris , variety  Olds’  Improved  Purple  Top,  New  Necklace 
and  Olds’  Long  White. 


Table  XL— Relative  Susceptibility  of  Different  Varieties  Brassica  cam- 
pestris to  Cystopus  candidus  from  Raphanus  sativus  Variety  Ne  Plus  Ultra. 


3 

o 

o 


Pertop 
OF  Re- 
FRI- 


3 

3 

3 

3 

3. 

1. 

2. 

1. 


Date. 


Host  plant. 


Source  of 
conidia. 


% 


• O 

o o 


■2; 


GERA- 

TION. 


a 

g S 
K H 


Dec.  4.. 
.Tan.  10. 
Jan.  10. 
Jan.  22. 
Jan.  22. 
Jan.  28. 
Sept.  23. 
Sept.  25. 


Olds’  improved  purple  top. 

New  neckless 

Olds’  large  white 

Olds’  large  white 

New  neckless 

Olds'  large  white 

Olds’  large  white 

Olds’  large  white 


Ne  Plus  Ultra. 
Ne  Plus  Ultra. 
Ne  Plus  Ultra. 
Ne  Plus  Ultra 
Ne  Plus  Ultra. 
Ne  Plus  Ultra. 
Ne  Plus  Ultra. 
Ne  Plus  Ultra. 


9 

9 

9 

9 

9 

3 

6 

3 


5 5 

5 24 
5 24 
5 26.5 
5 24.5 
5 20 
5 9.5 

5 3 


17 

10 

10 

12 

12 

12 

12 

9 


Results. 

Controls. 

<L 

Q 

No.  Coty  inf. 

Dec.  22. 

0 

Inf. 

Jan.  22. 

0 

Inf. 

Jan.  22. 

0, 

Inf. 

Feb  5.. 

0 

Inf. 

Feb.  5. . 

0 

Tnf. 

Feb.  15. 

0 

Inf. 

Oct.  8... 

0 

Tnf. 

Oct.  9. . . 

0 

Inf. 

62  Wisconsin  Experiment  Station. 

Summary  of  Table  XII 

Experiments  were  undertaken  to  determine  whether  Cyst  opus 
candidus  from  the  radish  could  be  made  to  grow  on  different 
varieties  of  cabbage  (Brassica  oleracea).  About  twenty  plants 
were  grown  in  each  three-inch  pot.  Fifteen  different  varieties 
of  cabbage  were  tested  from  January  10  to  March  13,  1910.  In- 
oculations were  made  in  the  usual  way  by  spraying  the  spores  on 
with  an  atomizer  and  placing  the  pots  containing  the  plants  in 
the  ice  box  under  a bell  jar  on  a wet  plate.  Three  tests  were 
made  on  each  variety,  so  that  at  least  sixty  plants  of  each  variety 
were  inoculated.  Four  of  the  varieties  tested  became  infected. 
All  Head  Early  showed  two  cotyledons  infected  out  of  a total 
of  sixty  plants  tested.  The  Volga  was  the  most  susceptible  to 
Cystopus  candidus.  Nine  cotyledons  became  infected  out  of  a 
total  of  sixty.  One  cotyledon  became  infected  out  of  sixty 
plants  of  Olds’  Selected  Early.  Of  the  sixty  plants  of  Olds’ 
Ballhead  inoculated,  two  cotyledons  were  infected.  In  all  the 
fifteen  varieties  of  cabbage  tested,  nine  hundred  plants  and  of 
course  nearly  twice  that  number  of  cotyledons  were  inoculated 
and  only  fourteen  cotyledons  became  infected.  The  results  show 
that  it  is  possible  to  infect  at  least  four  of  the  fifteen  varieties 
of  cabbage  tested  with  Cystopus  candidus  from  the  radish,  yet 
not  to  any  marked  extent. 

The  fungus  had  a more  marked  effect  on  the  cotyledons  of  the 
cabbage  than  on  those  of  the  normal  host,  the  radish.  The  in- 
fections found  were  always  on  sickly  looking  cotyledons  and  in 
two  or  three  days  after  the  pustules  appeared  the  cotyledons 
would  dry  up  and  drop  off.  In  the  radish  plants  used  as  con- 
trols the  cotyledons  lived  for  two  or  three  weeks  after  becoming 
infected.  This  would  mean  that  the  fungus  was  more  virulent 
on  the  cabbage,  and  immediately  killed  the  host  when  it  was 
able  to  establish  itself,  or  that  it  attacked  only  the  sickly,  weak 
cotyledons.  I am  inclined  to  believe  that  the  fungus  attacked 
all  of  the  cotyledons  alike,  but  the  further  development 
of  *the  fungus  was  overcome  by  the  host  in  all  the  cases  except 
in  the  few  infections  described  above.  This  is  of  course  a point 
that  needs  further  investigation  before  definite  information  can 
be  had. 

To  still  further  determine  the  susceptibility  of  different  varie- 
ties of  Brassica  oleracea  to  Cystopus  candidus  of  the  radish,  an- 


Table  XII. Relative  Susceptibility  of  Different  Varieties  of  Brassica  oleracea  to  Cystopus  candidus  from  Raphanus  . 

sativus,  Variety  Ne  Plus  Ultra. 


Experiments  on  Spore  Germination. 


63 


o g 
op 


>5 

-P 

,g  "H 

o a 

o'"1 


(O^HIMOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 


CNNONNNOO 


H<ooooo^^^Tt^Miftioinino^iDinin»oi222H 

P Sh’  fcJ  6h  Sh  G P P fr,-  P P Sh*  P ll  Sh*  P P Sh"  P P O ,Q  £ £5  b 5?  *”?  b b b "5  *5 

oaQaQaaQaaaaaflaaaaRaSiljj:  ^.2,2^2222222 


*5 

*©  £3 
2.0 

5? 


§3 

O 

£ 


• t-  !0  tD  t>-  tD  "O  <X>  ‘-D  tD  iO  CO  *.D  50  (O  PO  -*  35  t- t * - 


S 2 g g 2 s £ 2 g S 2 S S S S S 2 2 S 2 

SS33333333SS33333333  < 

“=3888888833. 

OOOOOOOOOOOOOOOOOOOO 

p a p p P 

JsssJsss^lsssIsssss 

OGCBC/JX^CCCCCC*  S®cB®SaQac!*®oQ® 


• ^ s-d 

*111 


SS3S38S88aS8asa%S888*,S®S8888888aa8a 


IIIIIIIIIIIiI!lllllllSS|l  i 


ppppBppppp2p£pBppBpBBpp2p;~ 


11 


a,ro  ap  w m 

SSWigag 

„ .2+>,s5a®>?a2®g 

p£ 322  *2  § * «2 ©2 
<!^«OOHOP3WHOWO 


*P 

3^ 


•d-d 


pp 


el 

P 33 

r£& 

%go22 

SmO®  g«®  P.2 
® "m  s^p'x^h  s 
^2^3^23^^ 

ogOPPPOHPP 


22g£ 

a*p  ; qG  hq3 

§l!t^s^lis 

gsgsSgHsSsgB^ 

rfocSwXrjoP— o^;  — P 
OPPPP^PO^PP^O 


ssssssssssssssssssss * ^xssssssssssss 


IC0WHWHHHH( 


Table  Xll.Continued.— Relative  Susceptibility  of  Different  Varieties  of  Brauica  ohracea  to  Cystoput  ctmdidm  from 

HGpha%ns lativve,  Vabieiy  Ne  Plus  I'lte/. 


64 


Wisconsin  Experiment  Station. 


IIS 


£ c 

. o 


it 


C * 

8 G 


o 5 


• * ird 
a.5- 


O 

J 8 


*H  «W  =1-  SH  «H  «W  S-i  <t-’  O-I  «i_*  o_‘  oj  ' -• 

ESC  c S3  C C 2 c a S3 

oooooooooooooooowoooooooooooo 

WQO^OT^TOmmMMMroOTOTMmMWMfQeoeoeofccococowci 

Hl'llll  5 S &&  & & 5 S S &S  &S  & S&iSssis 

S2SSSS22””S2coi:ocv:’<rCfCfCfCfCi«(«f«W’-'-^-iM(Mfccfl 


ooooo 


SSS^gggggggoooooooooooooooo 


%tf^iiiiiiiiiniiiiiiiniii 

mmmmrmiimm 


^■§ 

KK 


1? 


J=§ 

|5| 

S^J 


Ssigii 


C Q,  tn 


'gs-S'S'S-S 

a>  3 cs-o  a;  c3 

— t.  0)  0/ 

W £ P 3 
Sh  Q*  S 


1. 

■sl 


E« 

2X3 

of 


IS  !| 

. <D  . ^ 
:= 


i§§* 


'S  I5-2 


23  ;x3 
8.  “ 


SSo^'S.g'SS.?* 


ggS' 


jQ^W 


<L> 

« & 
rr  o 


22§  8^^  § Ssl  g 32*  § 822  §2  §2  32  o2  || 
OOfe«HE-iaQpOOD.COCSfi«OOHJOJOKOWWffl5 


“JD  *0  s©  CD  *© 


S^S88SSSS8SSSSSSgJJJasgs' 


b b b b f-  S-‘  «'  U t.'  in'  >s  >5  >.  it  bi  tl  L 

SiSdcsojQaaqipiftOQaaaaaQaQartacJspaQ 


Experiments  on  Spore  Germination. 


65 

other  series  of  experiments  was  made  from  April  13  to  August 
24,  1910.  The  fifteen  varities  of  cabbage  were  all  tested  again 
as  well  as  Kale,  Kohlrabi,  Brussel’s  Sprouts,  and  Cauliflower  as 
shown  in  Table  XII.  From  twenty  to  sixty  plants  were  inocu- 
ated  and  tested  as  described  above.  No  infections  were  obtained 
in  this  series  except  on  one  plant  of  Brussel’s  Sprouts.  One 
plant  became  infected  and  showed  five  pustules  on  its  twp  cotyle- 
dons, which  again  indicates  only  very  slight  susceptibility  in 
all  the  varieties  of  Brassica  oleracea  tested. 

Summary  of  Table  XIII 

The  mustards,  both  Brassica  nigm  and  Brassica  alba  were  next 
inoculated  with  spores  of  Cystopus  Candidas  from  the  radish. 
The  seedlings  were  grown  in  three-inch  pots  in  the  same  way  as 
described  for  the  experiments  on  the  turnips,  and  inoculated  by 
spraying  the  spores  on  with  an  atomizer.  The  plants  were  placed 
under  a bell  jar  on  a wet  earthen  plate  as  previously  described 
and  placed  in  the  ice  box  for  various  periods  of  time,  varying 
from  3 to  23%  hours. 

Six  cultures  of  three  plants  each  were  tested.  In  all,  eighteen 
plants  of  black  mustard  were  inoculated  with  Cystopus  Candidas 
from  the  radish,  variety  Ne  Plus  Ultra,  None  of  the  eighteen 
plants  became  infected.  A variety  of  white  mustard,  New  White 
Chinese,  was  tested  eight  times.  Each  time  using  three  plants 
in  the  same  manner  as  in  the  case  of  the  black  mustard.  Again 
no  infections  were  secured.  Further  thirteen  tests  of  three 
plants  each  or  a total  of  thirtynine  plants  of  white  mustard  were 
inoculated  with  Cystopus  Candidas  from  the  radish,  variety  Ne 
Plus  Ultra.  In  five  of  the  trials  infection  occurred;  three  coty- 
ledons and  six  leaves  became  infected.  The  infections  secured  on 
the  white  mustard  were  not  as  vigorous  as  those  secured  on  the 
controls  in  the  same  experiment.  The  susceptibility  of  white 
mustard  was  further  tested  in  the  spring  of  1910  on  April  11 
and  May  6.  On  April  11,  three  seven-inch  pots  containing  both 
radish  and  white  mustard  growing  together,  were  inoculated 
when  they  were  nine  days  old.  Infection  occurred  in  seven  to 
eight  days.  The  amount  of  infection  varied  from  10  to  40  per 
cent  of  the  cotyledons.  On  May  6,  eight  tests  were  made  in 
which  the  seedlings  were  grown  in  three-inch  pots.  The  number 
pf  plants  per  pot  varied  from  seven  to  forty  and  when  the  plants 


Table  XIII. — Susceptibility  of  Brassica  alba  and  Brassica  nigra  to  Cystopus  candidus  from  Baphanus  sativus, 

Variety  Ne  Plus  Ultra. 


66 


Wisconsin  Experiment  Station. 


og 

05 


G 22  22  22  S 2 ECSCEEGEEEEEEEEGECSEEEECG 


o-3 

■c  8 
(25 
£ 


OOOOOOOOOOOOOOOiMrirHiMeCO  oo'o'^o^o  00^5 

'<*  co  to  to  to  to  to  t-t-cccoiniftcitooooooodoaoooooooooooaoooooas 


— — — — — — — 550255’-<’-'’'J~0*-‘'H'",<’-fCOOOOOOO<:'1  s'1  ^ 


t—  in  in  m 


3 , . 

• g 23  T3 

o x»  o ® 


.42  23  -d 
o a o © 
!^4H  ^ b3 


iniflifliflwimoiflininiflioifl^icunioiniflKnomoopooooooo 


csrocococococoaiastotorocotorocococoeococoMoot-ao 


° o3 

P 

!§ 


SSSSSSIJSSSSSSS  3 3 3 3 3 3 

Cl  a.  CU  ^ Ph  Ph  CU  Ph  Ph  Ph  Cl  Cl  CL  0-  CL  Cl  52  P-i  CL  CL  Cl  £ ” — — — .2  -2 .22  42 .22 .2 
© a)  ® © ® <d  © a?  a>  <d  © © as  ® a>  © ® © ® a)  © 


B’B. 


•--I 

r23rOfG) 

sss 


llllllllllllllllilllllllllllllll 


42—  ©42  ©42  ©42  © ©fHXj'^xjSSxjSSSSSSSSSlSSSSSS 

CQ«£Cfi£CP^CC^^£^£ 


S5&S888S®®§S£§ga!i38S£-gi8£2®®®;0'0'0®5:;3;S 


o o o o o o o 
7777777QQ 


6--_S 


(rjHHHHHrtCOrtNH'VHr.MHHHHHF-, 


Experiments  on  Spore  Germination. 


67 


were  eleven  days  old  they  were  inoculated  with  conidia  from  the 
radish.  All  of  the  experiments  showed  infection  on  May  18. 
From  35  to  50  per  cent  of  the  total  number  of  cotyledons  were 
infected  as  shown  in  Table  XIII.  Only  a small  number  of 
leaves  became  infected  due  to  the  fact  that  not  many  of  the 
plants  had  leaves  when  they  were  inoculated.  The  results  in 
these  experiments  show  clearly  that  the  white  mustard  is  sus- 
ceptible to  the  cystopus  that  occurs  on  the  common  radish. 

Summary  of  Table  XIV 

During  the  spring  and  summer  of  1910,  various  other  hosts  of 
Cystopus  were  grown  from  seed  in  the  green  house  and  inocul- 
ated as  already  described.  The  plants  were  grown  in  three  inch 
pots  and  were  vigorous  and  healthy  when  inoculated.  In  every 
species,  at  least  two  separate  tests  were  made.  The  number  of 
plants  in  each  pot  varied  from  one  to  twenty.  If  the  plants  to 
be  inoculated  were  large  before  inoculated,  enough  were  removed 
to  allow  the  remainder  to  develop  normally.  Twenty-six  plants 
cf  Cap  sella  Bursa-past  oris,  nineteen  plants  of  Sisymbrium  offici- 
nale, twenty  plants  of  S.  altissimum , forty  plants  of  Lepidium 
sativum,  forty  plants  of  Nasturtium  officinale,  sixty  plants  of 
Brassica  nigra,  and  thirty  plants  of  Iberis  coronata  were  inocu- 
lated and  no  infections  were  observed.  Inoculations  on  radish 
were  used  as  controls  in  every  case  and  infections  were  always 
obtained.  Further  tests  must  be  made  with  these  plants  before 
positive  statements  can  be  made  as  to  their  susceptibility  to  the 
spores  from  Cystopus  from  the  radish. 

It  should  be  noted  that  a larger  percentage  of  infection  was 
obtained  in  the  spring  than  in  the  fall.  Thirteen  tests  were 
made  in  the  fall  of  1909  from  October  20  to  December  28  in 
which  thirtynine  plants  were  inoculated  and  only  three  cotyle- 
dons and  six  leaves  became  infected.  While  in  the  tests  in  the 
spring  from  10  to  50  per  cent  of  the  cotyledons  became  infected. 
The  difference  in  extent  of  infection  was  possibly  due  to  a differ- 
ence in  the  host  plants.  The  white  mustard  seedlings  grown  in 
the  fall  were  not  as  vigorous  as  those  obtained  in  the  spring. 


No.  cult,  tested. 


Wisconsin  Experiment  Station. 


08 


Table  XIV. — Susceptibility 


op  Other  Crucifers  to 
From  Radish. 


Cystopus  candidus 


Date. 


3 June  9. 
3 I June  9. 
May  23, 
Apr.  29. 
Apr.  29. 
Apr.  2 t. 
May  6. 
Au?.  21. 
Apr.  30. 


Host  Plant. 


Capsella  Bursa-pastorls 
Sisymbrium  officinale... 
Sisymorium  officinale. . . 

Lepidium  satiyum 

Brassica  nigra 

Capsella  Bursa-pastoris' 
Nasturium  officinale.... 

iberis  umbellatta 

Sisymbrium  altissimum. 


Source  of 
Conidia. 


§5  \l 

o 


Ne  Plus 
Ne  Plus 
Ne  Plus 
Ne  Plus 
Ne  Plus 
Ne  Plus 
Ne  Plus 
Ne  Pius 
Ne  Pius 


Ultra. 

Ultra. 

Ultra 

Ultra. 

Uitra. 

Ultra. 

Ultra. 

Ultra. 

Ultra. 


20 

20 

200 

12 

12 

12 

10 

20) 

20 


Period 
of  refri- 
ger- 
ation. 

Results. 

Con- 

trols 

Inf. 

Hours. 

Temper- 

ature. 

Date. 

Coty  Inf. 

8 

14 

June  24. 

0 

I Inf. 

8 

14 

June  24. 

o 

1 Inf. 

22.5 

17 

June  8. 

0 

Inf. 

27.5 

9 

May  25. 

0 

Inf. 

27.5 

9 

May  25. 

0 

Inf. 

27.5 

9 

May  25. 

0 

Inf. 

5 

10 

May  21. 

0 

Inf. 

14 

12 

Sep.  9. 

0 

Inf. 

6 

13 

May  13. 

0 

Inf. 

DISCUSSION  AND  CONCLUSION 
Germination  of  the  Conidia 

These  studies  with  various  species  of  Cystopus  and  other 
Oomycetes  have  shown  that  germination  of  the  conidia  is  con- 
trolled by  certain  factors  of  which  temperature  is  the  most  im- 
portant. Prevost  (1807:33)  in  his  studies  made  over 
a century  ago  states  that  at  a temperature  of  12°  to  16°  R.  Cys- 
topus  candidus  spores  sometimes  germinated  in  40  to  45  minutes, 
whereas  they  ordinarily  required  from  one  to  two  hours.  DeBary 
(1863:14)  found  the  conidia  of  Cystopus  to  germinate  at  tem- 
peratures ranging  from  5°  to  25°  C.  My  own  results  have  led 
me  to  conclude  that  chilling  the  spores  to  a relatively  low  tem- 
perature is  necessary  fo,r  the  most  vigorous  germination.  In  our 
preliminary  series  of  over  one  hundred  cultures  of  Cystopus 
spores  at  green-house  temperature,  22  to°  33°  C.  during  the  sum- 
mer, only  a very  low  percentage  of  germination  occurred.  In 
none  of  the  cultures  were  zoospores  observed  and  the  only  indi- 
cation of  germination  was  the  presence  of  an  occasional  empty 
sporange  which  may  possibly  have  been  empty  before  the  spores 
were  brought  into  the  laboratory. 

In  a later  series  (Table  II)  during  the  summer,  including 
fortyfive  cultures  chilled  with  controls  at  room  temperature,  it 


Experiments  on  Spore  Germination. 


69 


was  found  that  85  per  cent  of  the  chilled  cultures  germinated, 
whereas  none  of  the  cultures  kept  at  higher  temperatures  germ- 
inated. In  a third  series  (Table  V)  of  seventythree  cultures 
during  the  following  spring  where  about  one  third  were  held 
at  room  temperature  (i.  e.  above  20°  C.)  scanty  germination 
occurred  in  48%  of  the  cultures,  whereas  69%  of  the  cultures 
which  were  kept  at  a temperature  below  20°  C.  showed  abundant 
germination.  This  last  series  shows  that  germination  may 
occur  at  room  temperatures  and  above,  as  has  already  been 
pointed  out  by  DeBary  (1863:14),  Zalewski  (1883:215),  Biisgen 
(1882:22),  and  Eberhardt  (1904:614).  But  it  was  also  clear  that 
the  percentage  of  germination  was  much  increased  by  using 
lower  temperatures  as  was  further  shown  in  the  behavior  of  the 
stock  cultures  described' in  connection  with  Table  VIII.  The 
chilled  stock  cultures  became  heavily  infected  wdiile  the  controls 
not  chilled  showed  only  a low  percentage  of  infection  as  is 
conclusively  shown  by  referring  to  plates  I and  II. 

Two  things  are  clearly  indicated  from  our  results.  First,  and 
most  important.;  temperature  exercises  a marked  influence  upon 
germination ; second,  this  influence  wras  more  marked  with  spores 
obtained  in  the  late  summer  and  autumn  than  with  those  de- 
veloped in  the  spring.  We  interpret  this  latter  difference  as 
probably  due  to  the  greater  vigor  of  host  and  fungus  during  the 
spring  months.  Be  this  as  it  may,  it  was  at  all  seasons  evTdent 
that  comparatively  low  temperatures  were  necessary  to  induce 
strong  normal  germination. 

Various  media  were  used  for  germination  trials  with  spores 
of  Cystopus ; such  as  rain  water,  distilled  water,  tap  water,  ex- 
tracts of  the  host,  sugar  solutions  and  certain  nutrient  media. 
No  germination  was  obtained  in  any  medium  except  water  and 
no  marked  difference  was  noted  in  the  percentage  of  germina- 
tion obtained  whether  it  was  rain  water,  distilled  water  or  tap 
water  from  Lake  Mendota.  In  all  of  the  subsequent  tests  this 
tap  water  was  used.  Where  spores  were  immersed  in  a drop  of 
water,  the  condition  of  the  surrounding  atmosphere,  whether 
saturated  or  dry,  and  the  amount  of  evaporation  had  no  effect 
on  the  percentage  of  germination.  Other  physical  changes  in  the 
culture  containing  the  spores,  such  as  diffusion  of  the  drop  and 
changes  in  surface  tension,  were  of  no  consequence. 

No  attempt  has  been  made  to  determine  with  exactness  the 
optimum  temperature  for  germination.  Indeed,  the  variations 


70 


Wisconsin  Experiment  Station. 


associated  with  seasonal  and  host  conditions  as  just  noted  and 
with  maturity  of  the  spores  may  preclude  perfectly  definite  con- 
clusions upon  this  point.  It  was  at  least  strongly  suggested  by 
the  large  number  of  experiments  made  in  the  laboratory  and 
from  the  observations  made  out  doors  that  the  optimum  for 
normal  spores  produced  under  the  best  conditions  was  about 
10°  C. 

Results  as  to  the  maximum  temperature  of  germination  tend 
to  substantiate  DeBary,  who  found,  as  previously  noted,  that 
the  maximum  temperature  was  25°  C.  In  a series  of  over  one 
hundred  cultures  carried  on  in  the  greenhouse  during  the  months 
of  July  and  August  of  1909  at  temperatures  varying  from  22° 
to  33°  no  germination  was  obtained.  Again  in'  a later  series 
(Table  V)  in  the  spring  of  1910  scanty  germination  was  secured 
at  25°  C.  Although  these  experiments  were  not  planned  espe- 
cially to  test  this  point,  yet  they  show  that  the  maximum  tempe- 
rature of  germination  is  about  25°  C. 

DeBary  (1860:236)  concluded  that  the  minimum  temperature 
for  germination  was  5°  C.  My  results  show  that  the  conidia  of 
Cystopus  will  germinate  at  temperatures  below  5°  C.  Twelve 
cultures  were  made  and  laid  on  blocks  of  ice  in  the  ice  chamber 
of  the  ice  box  and  nine  of  the  cultures  germinated  in  4%  to  27 
hours.  The  temperature  of  the  cultures  was  between  0°  and  10° 
C.  In  view  of  these  facts  it  is  quite  clear  that  the  minimum  is 
very  near  zero. 

Not  only  do  the  conidia  germinate  at  low  temperatures  in  the 
laboratory  but  also  out  of  doors.  Observations  were  made  on 
seven  different  days  between  5 and  9 o’clock  A.  M.  in  the  au- 
tumn of  1910,  and  in  every  case  except  one,  zoospores  were  found 
on  the  infected  leaves  of  both  radish  and  salsify.  The 
minimum  early  morning  temperatures  on  the  days  when  obser- 
vations were  made,  varied  from  5 to  11  2-3°  C.  as  is  shown  in 
Table  VI.  DeBary  also  records  having  found  the  motile  zoo- 
spores in  the  morning  dew.  Since  the  motile  zoospores  were  thus 
found  in  the  morning  dew  by  both  DeBary  and  myself,  it  sug- 
gests that  the  conidia  germinate  in  the  coolest  part  of  the  day 
when  moisture  is  at  hand.  According  to  Salisbury  (1908:556) 
it  is  a well  established  fact  that  the  surface  of  the  earth  is  the 
coolest  at  about  sunrise,  a condition  that  leads  to  the  formation 
of  dew  and  thus  moisture  and  low  temperature  naturally  asso- 


Experiments  on  Spore  Germination. 


71 


elate  themselves  in  the  environment  of  the  fungus  and  may  well 
have  come  to  have  a correlated  influence  on  its  development. 

Somewhat  similar  conditions  have  been  reported  in  the  rusts 
by  Jaczewski  (1910:21),  who  found  that  in  the  cereal  rusts  of 
Russia,  both  the  uredospores  and  aecidiospores  germinated  in 
the  morning  when  the  foliage  was  wet  with  dew  and  the  tempera- 
ture was  low.  The  relation.  of  dew  to  the  asparagus  rust  has 
been  pointed  out  by  Smith  (1904:19)  in  California.  He  found 
that  the  rust  spreads  most  rapidly  when  heavy  dews  are  preva- 
lent. It  should  be  noted,  however,  that  Smith  (1904:19)  men- 
tions no  temperature  factor  as  especially  important. 

The  relation  of  light  to  the  germination  of  the  spores  of  Cys- 
topus  was  not  as  marked  as  it  has  been  reported  for  Plasmopara 
and  various  species  of  Phytophthora.  Farlow  (1875:319)  con- 
cluded from  his  studies  with  Plasmopara  viticola  and  Phytoph- 
thora infestans  that  the  conidia  germinated  better  in  the  dark 
than  in  the  light.  Coleman  (1910:59)  who  has  recently  studied 
Phytophthora  omnivora  states  that  light  is  a very  important 
stimulus  to  germination.  I have  found  in  Cystopus  that  the 
conidia  do  not  germinate  in  the  light  at  high  temperatures  and 
that  they  do  germinate  in  the  light  at  low  temperatures.  My 
conclusion  on  the  first  point  is  based  upon  seventeen  experiments 
tabulated  in  Table  III,  while  the  latter  conclusion  is  evident  from 
my  outdoor  observations  (Table  VI)  and  also  from  laboratory 
studies  not  tabulated.  I have  also  incidentally  tested  Phy- 
tophthora infestans  and  Plasmopara  viticola  as  to  the  relation 
of  light  to  germination  and  have  found  no  such  marked  differ- 
ence as  has  been  reported  by  Farlow  (1876:419). 

Zalewski  (1883  -.215)  concluded  that  the  time  of  the  year  had 
an  effect  on  the  time  required  for  the  germination  of  the  spores 
of  Cystopus.  He  found  that  the  time  required  for  germination 
in  the  summer  was  two  or  three  hours,  while  in  the  fall  it  re- 
quired from  one  to  three  days.  I have  found  that  during  August 
the  average  length  of  the  period  of  refrigeration  was  3%  hours ; 
September,  7% ; and  October,  9 hours.  My  results  show  without 
doubt  that  the  time  of  the  year  has  a direct  influence  on  the 
time  required  for  germination. 

It  may  well  be  due  to  the  different  host  reaction  on  the 
fungus  in  spring  and  fall.  And  again,  the  different  weather 
conditions  of  spring  and  fall,  may  have  a direct  influence  on 


Wisconsin  Experiment  Station. 


the  conidia.  The  cause  of  this  increase  in  time  required  for 
germination,  I do  not  know,  and  it  cannot  be  definitely  deter- 
mined, until  we  are  able  to  absolutely  control  all  of  the  factors 
influencing  the  host  and  fungus. 

A more  direct  comparison  of  my  results  with  those  of 
Zalewski  would  be  possible  if  we  knew  the  method  he  used  in 
Ins  germination  experiments  and  the  number  of  tests  made. 

In  none  of  my  experiments  did  the  conidia  of  Cystopus  germ- 
inate by  the  production  of  germ  tubes  as  described  by  Tulasne 
(1854:77)  and  Hoffmann  (1859:210).  Zoospores  were  always 
produced  when  the  conidia  germinated.  It  should  be  noted  that 
not  all  of  the  conidia  germinated.  Some  of  the  spores  were  dead 
or  immature. 


Eberhardt  (1904:614)  considers  the  proper  maturing  of  the 
spores  as  the  most  important  factor  in  securing  germination.  As 
ncted  above,  his  method  was  to  carefully  collect  infected  leaves 
with  unopened  pustules,  wrap  them  in  moist  cloth  and  place 
them  in  a damp  chamber  until  the  pustules  were  about  to  burst. 
With  these  precautions  Eberhardt  experienced  no  trouble  in 
germinating  the  conidia.  I have  never  found  it  necessary  with 
the  method  of  chilling  described  to  exercise  such  precaution.  To 
be  sure,  it  is  quite  necessary  in  spore  germination  to  have  a 
goodly  supply  of  ripe  spores,  but  in  Cystopus,  where  the  spores 
are  produced  acropetalously  and  borne  in  a pustule,  no  such 
precautions  were  required  in  order  to  secure  plenty  of  ripe 
spores.  Had  Eberhardt  been  studying  Phytophthora  infestans 
or  Plasmopara  vit'icola,  where  the  spores  are  borne  on  long,  much 
branched  conidiophores  standing  out  from  the  surface  of  the 
k af,  such  a procedure  might  have  been  more  important.  Conidi?« 
i i om  pustules  in  all  stages  of  development  have  been  used  and 
the  conidia  readily  germinated  when  chilled.  In  some  of  the 


experiments  described  above  and  in  many  of  my  preliminary 
experiments  (Seepage  34)  not  chilled,  conidia  from  pustules  just 
about  to  open  were  used  without  securing  germination.  In  only 
five  cases  does  Eberhardt  (1904:624)  record  the  data  of  his  ex- 
periments. In  two  of  his  tests  the  temperature  varied  from  2° 
to  17°  C.  In  another  test  the  spores  were  placed  in  water  at  6 


o’clock  in  the  afternoon  and  germination  was  observed  the  next 
morning  at  7 o’clock.  Under  ordinary  conditions,  the  room 
temperature  during  the  night  would  be  lower  than  in  the  day 


Experiments  on  Spore  Germination. 


73 


and  may  well  have  been  between  15°  and  20°  C.,  which  is 
sufficient  chilling  to  cause  germination. 

The  two  remaining  tests  were  made  on  June  6 and  August  8. 
No  record  is  made  of  the  temperature  conditions.  On  the  basis 
of  such  data  and  the  microscopical  examination  of  the 
ccnidia  from  pustules  just  opened  in  which  he  found 
the  conidia  swollen  and  bottlenecked,  Eberhardt  concludes  that 
germination  depends  upon  the  proper  maturing  of  the  conidia. 
In  view  of  the  fact  that,  in  two  of  the  tests  made  by  Eberhardt, 
the  temperature  was  much  below  17°  C.,  and  in  another  the 
temperature  was  that  of  night  time,  it  seems  to  me  probable 
that  temperature  may  have  been  a more  important  factor  in  Eber- 
hardt’s  experiments  than  he  realized. 

Since  I conclude  that  temperature  is  a controlling  factor 
in  spore  germination  of  Cystopus,  it  is  worth  while  to  make 
a comparison  with  results  obtained  in  the  case  of  other  fungi. 

In  the  Myxomycetes,  Jahn  (1905:489)  has  found  that  if  the 
spores  are  soaked  in  water  for  36  hours  and  then  allowed  to  dry 
out,  they  will  germinate  in  about  30  minutes  when  again  moist- 
ened. High  temperatures  for  short  periods  and  then  normal 
temperatures  tend  to  hasten  the  germination  of  the  spores  of 
Reticularia.  More  recently  Kusano  (1909:8)  has  shown  that  a 
weak  acid  solution  is  possibly  the  normal  stimulus  and  that  a 
temperature  below  20°  C.  retards  germination.  It  is  at  once 
apparent  that  in  the  Myxomycetes  thus  far  investigated,  there 
is  no  correlation  of  low  temperature  and  spore  germination.  In 
certain  of  the  fleshy  Basidiomycetes  investigated  by  Duggar 
(1901  :38)  and  Miss  Ferguson  (1902:16)  they  found  that  tem- 
perature changes  had  only  a slight  tendency  to  increase  the  per- 
centage of  germination.  In  the  rusts  or  parasitic  Basidiomy- 
cetes, on  the  other  hand,  the  relation  of  temperature  to  germina- 
tion is  more  marked,  as  is  evident  from  the  wmrk  of  Ericksson 
and  Henning  (1896:73)  and  Jaczewski  (1910:21).  The  former 
found  that  fresh  aecidiospores  sown  in  water  at  room  tempera- 
ture gave  a very  low  percentage  of  germination,  but  when  the 
spores  were  placed  on  blocks  of  ice  for  a while  and  then  returned 
to  water  of  room  temperature,  a much  higher  percentage  of 
germination  was  obtained.  It  should  be  pointed  out  in  this  con- 
nection, however,  that  the  above  investigators  made  too  few  ex- 
periments to  draw  definite  conclusions.  Their  results  arc  fuT" 


74 


Wisconsin  Experiment  Station. 


thermore  misleading  in  that  such  extreme  temperatures  were 
used.  The  germinations  obtained  by  Ericksson  and  Henning 
were  possibly  not  due  to  the  temperature  of  melting  ice,  but 
rather  to  the  slightly  higher  temperatures  obtained  after  the 
slides  were  removed  from  the  blocks  of  melting  ice.  In  similar 
experiments  performed  with  the  spores  of  Cystopus  this  has  been 
found  to  be  the  case.  The  observations  of  Jaczewski  (1910:21) 
further  substantiate  my  conclusions.  He  found  that  the  aecidio- 
spores  of  Puccinia  graminis  germinated  in  the  morning  dew  out- 
doors and  at  temperatures  considerably  below  normal  in  the 
laboratory.  The  uredospores  were  found  to  germinate  best  also 
at  temperatures  slightly  below  normal  (18°  C.). 

It  is  evident  from  the  above  facts  that  the  spores  of  the  sapro- 
phytic Basidiomycetes  and  parasitic  Basidiomyaetes  respond 
differently  to  temperature  at  the  time  of  germination  and  we 
would  naturally  expect  that  forms  so  different  in  habit  and  en- 
vironmental relations  would  respond  differently.  In  the  later 
cases,  it  is  purely  an  adaptation  to  environmental  conditions  in 
much  the  same  way  as  I have  found  it  to  be  in  the  Oomycetes, 
although  the  relation  is  less  marked  in  the  rusts  than  it  has 
been  found  to  be  in  Cystopus  and  various  other  Oomycetes.  In 
the  Ascomycetes  there  has  been  no  correlation  of  low  tempera- 
ture and  spore  germination  revealed  up  to  the  present  time 
but  too  little  is  known  of  the  factors  influencing  germination 
in  this  group  to  draw  any  conclusions.  In  the  Fungi  Imper- 
fecti,  on  the  other  hand,  also  little  studied  as  to  the  .factors 
influencing  spore  germination,  it  has  been  noted  in  one  instance 
that  low  temperature  has  a direct  relation  to  spore  germination. 
It  is  very  important  that  parasitic  fungi  belonging  to  the  two 
above  mentioned  groups  should  be  investigated  as  to  tempera- 
ture relations  because  of  their  direct  bearing  on  remedial  meas- 
ures. 

In  order  to  inoculate  various  hosts  with  Cystopus,  Eberhardt 
(1904:625)  germinated  the  spores  as  described  above  and  placed 
the  water  containing  the  zoospores  on  the  cotyledons  of  the  plants 
to  be  infected.  In  some  cases  the  plants  were  immersed  in  water 
containing  zoospores.  The  method  of  inoculating  plants  wTith 
the  conidia  of  Cystopus  that  has  been  used  in  my  experiments 
was  based  on  the  relation  of  chilling  to  germination  of  the  coni- 
dia. The  spores  were  placed  in  water  and  sprayed  on  the  plants 


Experiments  on  Spore  Germination.  75 

with  an  atomizer,  then  the  plants  were  covered  with  a bell  jar 
and  placed  in  the  ice  box  long  enough  to  insure  germination. 
The  method  I ,have  used  is  more  nearly  that  which  occurs  in  the 
normal  environment  of  the  fungus  than  that  used  by  Eberhardt. 

No  difference  in  the  susceptibility  of  the  cotyledons  and  leaves 
has  been  noted  in  any  of  my  infection  experiments,  although 
DeBary  (1863:24)  concluded  from  his  experiments  that  in  Cap- 
sella  and  Lepidium  the  cotyledons  only  were  susceptible  to  in- 
fection and  that  in  various  species  of  Brassica,  both  cotyledons 
and  leaves  were  susceptible  but  usually  only  the  cotyedons.  Still 
further  tests  were  made  as  to  the  susceptibility  of  the  leaves  of 
the  above  hosts.  Twentyfour  radish  plants  were  used,  two  in 
each  of  twelve  pots,  which  had  been  grown  in  the  greenhouse 
and  had  at  no  time  shown  any  infection.  After  these  had  lost 
their  cotyledons  they  were  inoculated.  Thirteen  days  later 
twenty  of  the  plants,  i.  e.,  all  but  four,  showed  leaf  infection  at 
many  points.  A pot  culture  of  at  least  fifty  white  mustard 
plants  having  lost  their  cotyledons  and  at  no  previous  time  show- 
ing infection  were  inoculated  and  every  plant  showed  infection 
on  at  least  several  of  its  leaves.  Five  plants  of  Capsella  in  blos- 
som were  inoculated  and  four  of  the  plants  became  infected,  de- 
veloping large  white  pustules  on  both  the  stems  and  young 
fruits.  We  have  never  tested  Lepidium  sativum  with  Cystopus 
from  the  same  host  but  have  succeeded  many  times  in  infecting 
the  leaves  with  Cystopus  from  Capsella.  The  same  care  was 
exercised  in  growing  the  twTo  named  hosts  free  from  infection 
as  was  noted  for  radish  and  white  mustard.  The  details  of  these 
experiments  are  given  in  an  earlier  part  of  this  paper.  There 
can  be  no  doubt  of  the  susceptibility  of  the  leaves  of  the  various 
hosts  described.  I have  noted,  however,  that  the  leaves  of  radish 
plants  about  to  blossom  or  in  blossom  seldom  become  infected 
and  when  infection  does  occur  a marked  hypertrophy  results. 
This  was  evident  from  stock  culture  J,  which  may  be  taken  as 
typical  of  the  nine  described  in  connection  with  Table  VII.  It  wTas 
started  September  29,  1909,  and  became  infected  October  6.  The 
pustules  until  March,  1910.  Then  the  stronger  of  these 
plants  sent  up  flowering  stalks,-  bearing  scattered  leaves  and  blos- 
plants  sent  up  owering  stalks,  bearing  scattered  leaves  and  blos- 
soms. From  this  time  on  the  fungus  development  on  the  basal 
leaves  started  to  disappear  and  the  upper  leaves  remained  prac- 
tically free  from  infection,  not  only  in  culture  J,  but  also  in  the 


76 


Wisconsin  Experiment  Station. 


other  eight  cultures  listed  in  Table  VII.  This  same  condition 
has  been  repeatedly  observed  on  plants  growing  outdoors,  and 
1 believe  that  the  leaves  of  the  radish  plants  are  not  less 
susceptible  at  the  time  flowers  are  developing  than  earlier,  hut 
that  the  decrease  in  extent  of  infection  is  due  to  less  moisture 
being  deposited  on  the  upper  leaves.  The  flowers  and  young 
fruits,  cn  the  other  hand,  may  become  the  seat  of  systemic 
infection  at  this  stage  of  the  host  plant,  in  which  case  oospores 
are  produced. 

With  the  method  of  infection  well  established  my  attention 
was  directed  to  determining  the  relative  susceptibility  of  the 
different  varieties  of  radish.  Twentytwo  varieties  were  found 
to  be  susceptible  with  no  marked  degree  of  variation.  Another 
species,  Raphanus  caudatus  (rat-tail  radish),  was  tested  to  de- 
termine whether  different  species  in  the  same  genus  were  sus- 
ceptible to  the  conidia  from  Raphanus  sativus  (common  radish). 
It  was  found  that  Raphanus  caudatus  was  readily  infected. 
Further  tests  were  made  with  the  conidia  of  Cyst  opus  Candidas 
from  radish  on  species  of  other  genera;  Brassica  rapa  (turnip), 
B.  campestris  (rutabaga),  B.  napus  (rape),  B.  nigra  (black 
mustard),  B.  oleracea  (varieties:  cauliflower,  kohlrabi,  and  kale), 
Capsella  Bursa-past  oris  (shephard’s  purse),  Lepidium  sativum 
(garden  cress),  L.  virginicum  (wild  pepper  grass),  Sisymbrium 
officinale  (hedge  mustard),  S.  altissimum  and  Iberis  umbellata 
( candy-tuft).  In  none  of  the  above  cases  did  infection  occur. 
Infection  was  secured  on  both  cotyledons  and  leaves  of  Brassica 
alba  (white  mustard)  and  on  the  cotyledons  of  Brassica  oleracea 
(four  varieties  of  cabbage).  My  results  show  that  it  is  possible 
to  inoculate  several  other  crucifers  with  the  spores  of  Cystcpus 
obtained  from  the  radish,  which  tends  to  preclude  the  possibility 
of  so  called  physiological  species  in  accordance  with  Eberhardt’s 
conclusions ; yet  it  may  well  be  that  limited  specialization  exists 
when  further  cross  inoculations  with  the  spores  from  other 
hosts  have  been  made.  Eberhardt  has  already  raised  the  ques- 
tion as  to  the  existence  of  a biological  form  on  each  of  the 
groups : Lepidium — Capsella — Arabis  and  Brassica — Sinapis 

Diplotaxis.  My  results  show  further  that  the  spores  of  Cystopus 
on  the  various  species  of  Raphanus  are  quite  limited  but  it 
may  be  that  Brassica  alba  serves  as  the  bridging  species.  These 
are  questions  that  can  be  fully  determined  only  by  a large 


Experiments  on  Spore  Germination.  * 77 

number  of  cross  inoculations  with  the  spores  from  various  hosts 
of  Cystopus. 

As  has  been  pointed  out,  the  infections  that  were  secured  on 
B 'i  us  sic  a alba  and  B.  oleracea  with  the  conidia  from  radish  dif- 
fered in  appearance  from  those  usually  occurring  on  the  radish. 
Ihe  infection  on  the  radish  is  vigorous,  causing  marked  hyper- 
trophy and  developing  large,  white,  plump  pustules.  On  the 
white  mustard  and  cabbage  this  was  not  the  case.  No  hyper- 
trophy occurred  and  the  pustules  were  small,  showing  none  of 
the  signs  of  vigor  evident  on  the  radish.  Not  only  was  there  a 
marked  difference  in  the  appearance  of  the  fungus  pustules  on 
the  hosts  in  question  but  also  in  the  effect  upon  them.  The  fun- 
gus killed  the  host  tissues  very  much  faster  on  the  white  mustard 
and  cabbage  than  on  the  radish.  A possible  explanation  of  these 
results  would  be  that  the  infection  of  the  white  mustard  and 
cabbage  occurs  only  in  the  most  vigorous  cotyledons;  that  in 
these  the  fungus  is  able  to  overcome  the  host  cells  and  persist  in 
only  a few  cases  and  that  in  such,  the  host  cells  when  overcome 
die  immediately. 

In  my  observations,  plants  infected  with  aphids  or  thrips 
seem  to  be  quite  immune  to  Cystopus.  At  no  time  was  I able  to 
get  infection  on  a plant  that  was  badly  infected  with  insects. 
Reed  (1907 :381)  also  found  that  it  was  quite  impossible  to 
infect  grain  seedlings  with  mildew  that  were  already  infected 
with  thrips.  This  was  more  evident  in  the  case  of  wild  plants 
such  as  Capsella,  Lepidium  and  Sisymbrium  than  in  the  case  of 
such  cultivated  plants  as  radishes  and  mustards.  The  lack  of 
infection  can  not  be  attributed  to  the  aphids  eating  the  spores, 
since  some  of  the  plants  were  fumigated,  killing  the  insects  and 
then  inoculated,  with  similar  results.  These  facts  lend  support 
to  Cook’s  (1911:624)  view  that  plants  injured  by  plant  or  ani- 
mal parasites  develop  an  excess  of  tanin  which  causes  more  or 
less  immunity.  Not  only  was  it  quite  impossible  to  infect  plants 
attacked  by  insects,  but  likewise,  plants  that  showed  signs  of  not 
being  vigorous  and  healthy  from  other  causes.  It  was  also  im- 
possible to  infect  wild  and  cultivated  seedlings  that  showed  yel- 
lowing of  the  cotyledons  and  first  true  leaves.  This  was  also 
true  of  the  more  mature  plants.  If  for  any  reason  a stock  cul- 
ture of  radishes  showed  signs  of  not  being  healthy  and  vigorous 
the  extent  of  infection  was  at  once  reduced.  As  has  been 


78 


Wisconsin  Experiment  Station. 


stated  earlier,  Eberhardt  believed  that  the  various  hosts  of 
Cystopus  do  not  at  all  stages  of  development  show  the  same 
susceptibility  for  the  fungus.  Nowhere  does  Eberhardt  have 
any  data  to  substantiate  this  conclusion,  nor  has  he  taken  into 
consideration  host  abnormalities  as  a factor  influencing  the 
question  of  susceptibility.  Prom  my  results  it  is  at  least  very 
evident  that  Cystopus  reacts  differently  to  healthy  and  sickly 
plants  respectively. 

It  is  impossible  to  infect  Capsella  Bursa-past  oris,  Lepidium 
virginicum,  or  Sisymbrium  officinale  when  the  plants  are  not 
vigorous  and  healthy.  Many  attempts  were  made  during  the 
fall  of  1909  to  infect  Sisymbrium  officinale  with  Cystopus  can- 
didus  from  the  same  host  but  the  infections  were  very  scanty. 
Out  of  the  fourteen  experiments  on  fiftysix  plants,  only  eight 
plants  became  infected.  I attribute  this  to  the  weakness  of  the 
plants  that  were  grown  at  that  time.  In  every  case,  it  was  the 
largest  and  healthiest  looking  plants  in  the  lot  which  took  the 
disease.  Although  I have  not  succeeded  in  proving  entirely  to 
my  own  satisfaction  that  the  extent  of  infection  is  dependent 
upon  the  vitality  of  the  host ; yet  it  seems  highly  probable  that 
this  is  the  case.  Reed  (1907  :381)  has  fully  described  a similar 
relation  between  the  host  and  fungus  in  the  grain  mildews. 
Since  there  is  this  evidence  in  both  the  mildews  and  white  rusts 
that  sickly  hosts  do  not  readily  become  infected,  in  testing  a 
species  for  socalled  physiological  species,  all  possible  care  should 
be  exercised  in  cases  where  plants  are  used  as  hosts  that  are  at 
all  difficult  to  grow.  Failure  to  infect  may  be  due  to  weakness  of 
the  host  plant. 


Experiments  on  Spore  Germination. 


1 9 


SUMMARY 

The  studies  outlined  in  the  preceding  pages  were  carried  on 
chiefly  with  Cystopus  candidus  as  it  occurs  on  the  common  radish, 
Baphanus  sativus.  The  leading  problems  considered  are:  Con 

ditions  influencing  germination  of  the  conidia;  conditions  in- 
fluencing infection ; and,  the  occurrence  of  so-called  physiological 
species  of  Cystopus  candidus  on  the  various  crucifers. 

Germination  of  Conidia 

When  the  conidia  are  placed  in  water  they  germinate  better 
a strikingly  low  than  at  high  temperatures.  The  optimum  was 
not  definitely  determined,  but  the  results  tend  to  show  that  it 
was  10°  C.  The  minimum  temperature  of  germination  was  very 
near  zero,  while  the  maximum  was,  as  DeBary  has  shown,  about 

25°  C.  ' ; ! | :■ 

It  was  found  that  water  is  the  most  favorable  medium  for 
germination.  No  germination  was  obtained  on  various  nutritive 
culture  media. 

The  time  required  from  the  immersion  of  the  conidia  to  the 
escape  of  the  zoospores  usually  varied  from  two  to  ten  hours. 
The  shortest  period  in  which  such  germination  was  observed  was 
45  minutes. 

Environmental  factors,  season  and  host  vitality,  seemed  to  in- 
fluence the  time  required  for  the  spores  to  germinate.  It  was 
strongly  suggested  that  the  time  required  in  spring  and  summer 
is  shorter  than  in  the  late  fall  and  winter. 

No  difference  was  observed  in  the  time  or  percentage  of  germ- 
ination which  occurred  in  light  as  compared  with  darkness. 

Spores  obtained  from  leaves  after  a killing  frost  germinated. 

Such  factors  as  evaporation,  surface  tension,  and  diffusion 
of  the  drop  containing  the  conidia  did  not  influence  the  percent- 
age of  germination.  The  conidia  germinated  as  readily  in  a non- 
saturated  as  in  a saturated  atmosphere. 

/ 

Conditions  of  Infection 

Chilling  was  also  found  to  have  a very  marked  effect  on  the 
degree  of  infection  secured,  as  can  be  seen  by  referring  to  plates 
I to  X.  Ninetyfive  per  cent  of  the  seedlings  chilled  became  in- 


80 


Wisconsin  Experiment  Station. 


fected  while  the  controls  not  chilled  usually  showed  less  than 
‘3  per  cent  of  infection  and  never  more  than  15  per  cent.  This 
difference  in  extent  of  infection.  I believe  was  due  to  the  in- 
creased percentage  of  spore  germination.  It  should  be  noted,, 
however,  that  the  chilling  process  may  have  had  some  effect  on 
the  host,  possibly  making  it  more  susceptible.  This  is  a point 
that  needs  further  investigation. 

the  favorable  effect  of  chilling  on  the  conidia  of  Cystopus  is 
plainly  an  adaptation  to  the  environment  of  the  fungus.  The 
spread  of  a fungus  by  zoospore  infection  is  directly  dependent 
upon  the  presence  of  water  on  the  foliage  of  the  host.  DeBary 
found  the  motile  zoospores  of  Cystopus  in  the  dew  drops  in  the 
morning  on  the  host  plant  and  I have  often  made  the  same  ob- 
servation. The  fall  in  temperature  which  leads  to  the  deposition 
of  dew  and  thus  provides  a medium  in  which  the  zoospores  may 
develop  serves  at  the  same  time  as  the  necessary  stimulus  to  the 
germination  of  the  conidia. 

The  results  obtained  suggest  that  a close  relation  exists  be- 
tween host  vigor  and  susceptibility  in  that  healthy  plants  are 
more  susceptible  than  sickly  or  abnormal  ones. 

No  marked  difference  in  the  susceptibility  of  leaves  and  cotyle-  ; 
dons  of  the  radish,  shepherd’s  purse,  white  mustard  and  gar-  : 
den  cress  was  observed.  . ] 

So-called  Physiological  Species 

i 

Repeated  infection  experiments  were  made  using  conidia  of 
Cystopus  candidus  from  the  common  radish,  Raphanus  sativus. 
upon  this  same  and  other  cruciferous  hosts  to  learn  whether  ; 
there  is  any  difference  in  susceptibility. 

A large  number  of  experiments  were  made  testing  the  suscep-  j 
tibility  of  twenty  two  different  varieties  of  radish,  and  it  was  \ 
found  that  no  marked  difference  in  their  susceptibility  existed.  j 
It  was  also  readily  possible  to  infect  Raphanus  candatus  with  the  * 
conidia  from  Raphanus  sativus  which  shows  that  species  of  the 
sii me  genus  are  susceptible  to  the  form  of  Cystopus  that  occurs 
on  the  common  radish. 

Species  of  crucifers  from  other  genera  known  to  be  hosts  of 
the  white  rust  were  investigated  as  to  their  susceptibility  to 
Cystopus  candidus  from  the  common  radish.  Infection  was  se- 
cured on  the  white  mustard,  Brassica  alba , and  cabbage,  Bras-  5 


Experiments  on  Spore  Germination. 


81 


sica  oleracea.  At  no  time  was  it  possible  to  infect  more  than  50 
per  cent  of  the  cotyledons  or  leaves  of  white  mustard  which  were 
inoculated.  With  the  cabbage,  it  was  even  more  difficult  to 
secure  infection,  although  fifteen  varieties  weye  tested.  Less 
than  1 per  cent  of  the  plants  inoculated  became  infected. 

No  infection  could  be  secured  on  any  of  the  other  crucifers 
tested.  These  included  turnip,  Brassica  rapa,  ten  varieties; 
black  mustard,  B.  mg\m,  rutabaga,  B.  campestris,  three  varie- 
ties; shepherd ’s  purse,  Capsella  Bursa-past  oris;  garden  cress, 
Lepidium  sativum;  wild  pepper  grass,  Lepidium  virginicum; 
hedge  mustard,  two  species  Sisymbrium  officinale  and  S.  altis- 
simum;  candy  tuft,  Iberis  umbellata;  water  cress,  Nasturtium 
officinale , and  wall  flower,  Cheiranthus  cheiri. 


LITERATURE  CITED 

1807.  Prevost,  B. : Memoire  sur  la  Cause  immediate  de  la  Carie 
ou  Charbon  des  Bles,  etc.,  pp.  33-35. 

1854.  Tulasne,  L.  R. : 2nd  memoire  les  Uredinees  et  les  Ustil- 
aginees.  Ann.  Sci.  Nat.  Bot.  Series  IV  1&2:77. 

1859.  Hoffmann,  II.:  Ueber  Pilzkeimungen.  Bot.  Ztg.  17:210 

1860.  DeBary,  A. : La  formation  de  zoospores.  Ann.  Sci. 

Nat.  Bet,  series  IV  13&1 4:236. 

1863.  DeBary,  A. : Recherches  sur  le  developpement  de  quel- 

ques  champignons  parasites.  Ann.  Sci.  Nat.  Bot., 
series  IV.  2 0:14. 

1873.  Wiesner,  J. : Sitzber  Akad.  Wiss.  (Vienna)  Math.  Phys. 

Kl.  68,  1.  (Abstract  from  DeBary  Morphology  of 
Fungi,  p 349.) 

1875.  Farldw,  W.  G. : The  Potato  Rot.  Bui.  Bussey  Inst.  Part 

IV  p.  319. 

1876.  Farlow,  W.  G. : The  American  Grape  Vine  Mildew. 

Bui.  Bussey  Inst.  Art.  I.  p.  419. 

1882.  Busgen,  M. : Die  Entwicklung  der  Phycomycetensporan- 

gien.  Jahrb.  Wiss.  Bot.  [Pringsheim]  13:22. 

1883.  Zalewski,  A. : Ueber  Sporenabschnurung  und  Sporenab- 

fallen  bed  den  Pilzen.  Flora  66  :251. 

J883.  Zalewski,  A. : Zur  Kenntniss  der  Gattung  Cystopus, 
Bot.  Centbl.  15:215. 


82 


Wisconsin  Experiment  Station. 


1886.  Scribner,  F.  L. : The  Downy  Mildew.  Bot.  Diy.  U.  S. 

Dept.  Agri.  Bnl.  2,  p.  10. 

1893.  Viala,  P. : Les  maladies  de  la  Vigne,  p.  93. 

1895.  Eriksson,  J. : Ueber  die  Fordernng  der  Pilzensporen- 

keimnng  durch  Kalte.  Centbl.  Bakt.  (etc.).  2 Abt. 

1 :557. 

1896.  Eriksson  and  Henning. : Die  Getreideroste.  Stockholm. 

p.  73. 

1901.  Ludi,  R. : Beitrage  zur  Kenntniss  der  Chytridiaceen. 
Hedwigia,  40:1-44. 

1901.  Duggar,  B.  M. : Physiological  Studies  with  Reference 

to  the  Germination  of  Certain  Fungous  Spores.  Bot. 
Gaz.  31:38-66. 

1902.  Ward,  IT.  M. : On  Relation  between  Host  and  Parasite 

in  the  Bromes  and  their  Brown  Rusts.  Ann.  Bot. 
16:265. 

1902.  Ferguson,  Margaret  C. : Germination  of  the  Spores  of 
Agaricus  eumpestris  and  Other  Basidiomycetous  Fungi. 
U.  S.  Dept.  Agri.  Bur.  Plant  Indus.  Bui.  16:16. 
1903-4.  Eberhardt,  Albert : Zur  Biologie  von  Cystopus  candi- 
dus.  Centbl.  Bakt.,  (etc.).  Abt.  2.  1 0 :655-656. 

1904.  Eberhardt,  Albert:  Contribution  a Petude  de  Cystopus  * 
candidus.  Centbl.  Bkt.  (etc.).  Abt.  2.  12:614-631  and 
71T-727.  : 

1904.  Clinton,  G.  P. : Downy  Mildew,  or  Blight  of  Musk  i 

Melons  and  Cucumbers.  Conn.  (New  Haven)  Agri.  j 

Exp.  Sta.  Rpt.  Part  4.  Botanist  Rpt.  p.  338. 

1905.  Jahn,  E. : Myxomyeetenstudien.  Ber.  Deutsch.  Bot. 

Gesell.  Abt.  2.  23:489. 

1906.  Reed,  Geo.  M. : Infection  Experiments  with  Erysiphe  > 

graminis.  Wis.  Acad,  of  Sci.,  Arts  and  Letters.  Part  1,  | 
15 :135.  j 

1907.  Reed,  Geo.  M. : Infection  Experiments  with  Erysiphe  j 

Cichoracearum  DC.,  Bui.  of  Wis.  Univ.  No.  250,.  Sc.  ' 
series  3,  No.  2.  3 :381. 

1904.  Smith,  R.  E. : The  Water  Relation  of  Puccinia  Asparagi. 

Bot.  Gaz.  38  :19. 


Experiments  on  Spore  Germination. 


83 


1908.  Clinton,  G.  P. : Artificial  Cultures  of  Phytophthora 
with  Especial  Reference  to  Oospores.  Conn.  (New 
Haven)  Agri.  Exp.  Sta.  Rpt.  Part  4.  Botanist  Rpt.  p. 

904.  : : I ■ , 1 1!  »] 

1908.  Salisbury,  R.  D. : Physiography,  p.  356. 

1910.  Jaczewski,  A. : Studien  uber  das  Verhalten  des  Schwarz- 

rostes  des  Getreides  in  Russland.  Ztschr.  Planzen- 
krank.  20:21. 

1911.  Cook,  M. : Protective  Enzymes.  Science,  n.  s,  33:625. 


84 


Wisconsin  Experiment  Station. 


DESCRIPTION  OF  PLATES 

The  following  plates  are  all  . from  photographs  of  radish 
plants  grown  in  six  inch  pots,  taken  from  above.  In  some 
cases  the  entire  culture  is  shown  with  a slight  reduction.  In 
the  rest  only  a portion  of  the  culture  is  shown,  but  so  selected 
as  to  be  fairly  representative.  The  plates  illustrate  the  advan- 
tage of  chilling  in  securing  optimum  spore  germination  and 
favorable  conditions  for  infection  with  Cyst  opus  candidus.  The 
seedlings  were  inoculated,  covered  with  bell  jars  and  either- 
placed  in  an  ice  box  or  kept  at  room  temperature. 

I.  Radish.  Var.— Ne  Plus  Ultra.  Sowed  May  16.  Inoculated 
May  26.  Infected  -June  2.  Photographed  June  6.  This 
culture  was  chilled. 

II.  Control.  Radish.  Var.— Ne  Plus  Ultra.  Sowed  May  16. 
Inoculated  May  26.  Infected  June  2.  Photographed  June  6. 
This  culture  was  not  chilled. 

III.  Radish.  Var.— Ne  Plus  Ultra.  Sowed  Nov.  1.  Inoculat- 
ed Nov.  8.  Infected  Nov.  15.  Photographed  Nov.  17.  This 
eulture  was  chilled. 

IV.  Control.  Radish.  Var.— Ne  Plus  Ultra.  Sowed  Nov.  1. 
Inoculated  Ncv.  8.  Infected  Nov.  15.  Photographed  Nov.  17. 
This  culture  was  not  chilled. 

V.  Same  culture  as  shown  in  III.  Photographed  twelve 
days  later,  Nov.  29. 

VI.  Same  culture  as  shown  in  IV.  Photographed  twelve 
(days  later,  Nov.  29. 

VII.  Radish.  Var.— Ne  Plus  Ultra.  Sowed  Nov.  6.  Inoculat- 
ed Dec.  3.  Infected  Dec.  12.  Photographed  Jan.  1,  1910.  Cul- 
tures at  right  of  page  chilled ; at  left  of  page  controls,  not  chilled. 

VIII.  Radish.  Var. — Triumph.  Sowed  Dec.  12,  1909. 

Inoculated  Jan.  3,  ’10.  Infected  Jan.  12,  1910.  Photographed 
Jan.  18,  1910.  Upper  culture  chilled.  Lower  culture  control, 
not  chilled. 

IX.  Radish.  Var.— Ne  Plus  Ultra.  Sowed  May  26,  1910. 
Inoculated  June  6.  Inf.  June  12.  Photographed  June  15. 
This  culture  was  chilled. 

X.  Control-Radish.  Var.— Ne  Plus  Ultra.  Sowed  May 
26,1910.  Inoculated  June  6.  Inf.  June  14.  Photographed  June 
15.  This  culture  was  not  chilled. 


Experiments  on  Spore  Germination, 


85 


I.— Radish  seedlings  inoculated  with  Cystopus,  chilled.  (Compare  with  II.) 


Experiments  on  Spore  Germination 


86 


II. — Radish  seedlings  inoculated  with  Cystopus,  not  chilled.  (Compare  with  I.) 


4 


j 

,3 

( 

. 


Experiments  on  Spore  Germination. 


87 


III —Radish  seedlings  inoculated  with  Cystopus;  chilled. 


IV.— Radish  seedlings  inoculated  with  Cystopus;  control  not  chilled. 


% 


% 


<3^ 

e 


% 


V* 


Experiments  on  Spore  Germination. 


88 


V.— Radish  seedlings  inoculated  with  Cystopus;  chilled. 


VI.— Radish  seedlings  inoculated  with  Cystopus;  control  not  chilled. 


Experiments  on  Spore  Germination. 


89 


ATI.— Four  small  cultures  of  radish  seedlings;  two  at  right  of  page,  chilled;  two, 
controls,  at  left  of  page,  not  chilled. 


\ 


\ 

<P 


Experiments  on  Spore  Germination. 


90 


Larger  radish  plants  inoculated  with  Cystopus;  chilled. 


Vm._Large  radish  plants  inoculated  with  Cystopus;  control  not  chilled. 


Experiments  on  Spore  Germination, 


91 


IX. — Radish  seedlings  inoculated  with  Cystopus;  chilled. 


X.— Radish  seedlings  inoculated  with  Cystopus;  control  not  chilled. 


’l^At'OUwclv  In iidXvu 

The  Place  of  Economics  in  Agricultural 
Education  and  Research 


H.  C.  TAYLOR 

To  direct  any  business  intelligently  the  manager  must  have 
a clear  vision  of  the  causes  which  affect  the  results  of  his  activi- 
ties. The  purpose  in  applying  scientific  methods  in  the  study 
of  agriculture  is  to  gain  knowledge  of  the  forces  and  conditions 
with  which  the  farmer  has  to  deal.  Some  of  these  forces  are 
physical,  some  are  biological,  and  some  are  economic. 

The  physical  and  biological  sciences,,  when  applied  to  agricul- 
ture, have  to  do  with  the  harmonious  adjustment  of  the  relations 
between  the  useful  forms  of  plant  and  animal  life  and  their  phy- 
sical and  biological  environment.  Economics  when  applied  to 
agriculture  has  to  do  with  the  harmonious  adjustment  of  the 
relations  between  useful  forms  of  plant  and  animal  productions 
and  the  human  environment ; also  between  the  various  people 
who  participate  in  the  production,  transportation  and  marketing 
of*  farm  products.  It  is  the  function  of  the  physical  and  bio- 
logical sciences  to  make  clear  the  physical  and  biological  forces 
with  which  the  farmer  has  to  deal.  This  will  show  what  it  is 
possible  to  produce  and  the  various  possible  methods  of  produc- 
ing each  article.  It  is  the  function  of  economics  to  make  clear 
the  economic  forces  with  which  the  farmer  has  to  deal  and  to 
develop  methods  of  ascertaining  what  to  produce  and  how  to  pro- 
duce it  in  order  to  secure  maximum  net  profits  for  the  farmer 
and  maximum  wellbeing  for  the  nation. 

Economics  and  the  Problem  of  Crop  Selection 

From  the  standpoint  of  soil  and  climate  a given  locality  may 
be  well  suited  to  the  production  of  a given  product,  and  yet 
when  the  test  of  maximum  net  profits  to  the  farmer  or  the  test 


94 


Wisconsin  Experiment  Station. 


of  economy  of  energy  in  the  satisfaction  of  the  wants  of  the 
community  for  this  product  is  applied,  its  production  may  be 
found  unprofitable.  This  raises  the  question,  “What  are  some 
of  the  economic  forces  and  conditions  which  have  to  be  taken 
into  account  in  addition  to  the  physical  and  biological  factors 
in  determining  what  to  produce  in  a given  locality?” 

Opportunity  for  marketing  the  product  suggests  itself  at 
once  as  an  important  item  to  be  considered.  The  abundance  or 
scarcity  of  labor,  and  the  abundance  or  scarcity  of  capital  in  a 
given  locality,  in  comparison  with  other  localities  where  the  soil 
and  climate  are  equally  good,  become  important  determining 
factors.  Again,  of  two  localities  with  soil  and  climate  equally 
well  suited  to  the  production  of  a given  crop,  one  locality  may 
be  suited  also  to  another  crop  which  requires  the  attention  of 
the  farmer  at  the  same  time  of  year  and  which  is  a more  profit- 
able crop. 

To  illustrate  the  way  in  which  economic  forces  need  be  taken 
into  account  in  determining  which  crops  to  grow,  take, 
for  example,  the  beet-sugar  industry.  Sugar  being  desired, 
man  has  put  forth  efforts  to  secure  a supply.  The  problems  of 
securing  this  supply  brought  into  requisition  such  plants  as  by 
nature  contain  sugar.  Among  other  plants  a variety  of  beet 
was  found  to  contain  sugar.  The  news  went  forth  that  the  sugar 
supply  could  be  secured  from  the  beet.  Every  intelligent  farmer 
asked  himself  the  question,  “Why  should  I not  produce  sugar 
beets?” 

The  physical  and  biological  sciences  were  at  once  brought  to 
bear  upon  this  problem  to  ascertain  the  soil  and  climatic  condi- 
tions wThich  are  best  suited  to  the  growth  of  the  sugar  beet. 
Geology,  meteorology,  physics,  chemistry,  entomology,  plant  phy- 
siology, plant  breeding,  bacteriology,  etc.,  made  their  contribu- 
tion to  the  farmer’s  knowledge  of  the  regions  in  which  beets 
thrive,  the  varieties  of  beets  containing  the  highest  percentage 
of  sugar,  the  methods  of  cultivation  which  will  best  adjust  the 
soil  to  plant  growth,  the  methods  of  protecting  the  plant  from 
vegetable  and  animal  parasites,  etc.  Tables  were  published 
showing  the  percentage  of  sugar  found  in  the  beets  from  dif- 
ferent seeds  on  the  same  soil,  and  from  the  same  kinds  of  seeds 
on  different  soils  and  under  different  climatic  conditions.  Maps 
were  made  showing  the  regions  where  the  climate  was  suitable 


Economics  in  Agricultural  Education  and  Research.  95 

for  sugar  beet  culture.  Soils  were  surveyed  with  a view  to 
finding  the  land  best  suited  to  the  sugar  beet. 

With  this  knowledge,  which  appeared  all  sufficient  to  the 
minds  of  many  experiment  station  men,  beet  culture  was  advo- 
cated without  asking  the  question,  “Where  is  the  beet  sugar 
industry  likely  to  prove . profitable  ? ” 

The  profitable  introduction  of  the  sugar  beet  in  any  given 
locality  where  soil  and  climate  are  suitable  depends  upon  the 
relative  profitableness  of  this  crop  when  compared  with  other 
crops  occupying  the  same  place  in  the  rotation  and  requiring 
the  attention  of  the  farmer  at  the  same  time  of  year.  For  ex- 
ample,  in  the  sugar  beet  regions  of  Germany,  Indian  corn  does 
not  thrive,  and  in  the  absence  of  competing  crops  which 
are  very  . profitable,  beets  stand  a,  better  chance  (other 
things  being  the  same)  than  in  the  corn  belt  of  the 
United  States,  where  corn  is  a very  profitable  competitor  of 
beets.  In  those  parts  of  Germany  where  the  beet  sugar  industry 
prospers,  beets  have  only  to  prove  as  profitable  as  potatoes,  root 
crops  grown  for  fodder,  or  a bare  fallow,  in  order  to  find  a profit- 
able place  in  the  field  system,  whereas  in  the  corn  belt  of  the 
United  States  beets  must  prove  as  profitable  as  corn  or  give 
place  to  if. 

The  relative  profitableness  of  corn  and  sugar  beets  can  be 
ascertained  by  a system  of  records  which  will  show  all  the  ele- 
ments  of  costs  and  receipts  of  the  two  crops  and  their  influence 
upon  the  profitableness  of  the  other  enterprises  of  the  farm.  In 
the  consideration  of  the  relative  profitableness  of  corn  and  beets, 
account  must  be  taken  of  the.  difference  in  the  acreage  of  each 
crop  a farmer  can  manage.  It  is  well  known  that  a farmer  can 
grow  more  acres  of  corn  than  of  beets.  It  is  a mistake  there- 
fore to  compare  profits  per  acre  and  to  stop  there.  Profit  per 
acre  must  be  multiplied  by  the  number  of  acres  the  farmer  can 
handle.  Furthermore,  the  way  in  which  the  corn  or  the  beets 
complement  the  other  crops  with  respect  to  utilization  of  labor 
and  equipment  should  be  considered.  It  is  always  desirable  to 
have  an  even  and  'continuous  demand  for  man  and  horse  labor. 
It  is  also  important  to  consider  the  profitableness  of  the  other  en- 
terprises such  as  dairying,  cattle  feeding,  or  hog  feeding,  which 
may  be  based  upon  the  corn  crop. 

But  after  all  these  calculations  have  been  made  and  the  beets 
have  been  found  profitable  to  the  farmers  of  a region,  the  further 


96 


AyiscoNSiN  Experiment  Station. 


questions  may  well  be  asked,  “What  conditions  exist,  such  as 
bounties,  tariffs  on  imports,  etc.,  which  maintain  the  price  of 
sugar  at  an  artificial  level  in  this  country?  Would  the  sugar 
beet  be  profitable  to  the  farmer  if  the  price  of  sugar  were 
lowered  to  the  level  of  the  open  market  of  the  world?  If  not, 
is  it  profitable  to  the  people  of  the  nation  as  a whole  to  have 
sugar  produced  from  the  beets  V’  These  are  economic  questions 
which  should  be  faced  squarely  and  answered  as  accurately  as 
possible. 

Some  crops  can  be  grown  over  a wide  area.  Others  are  more 
limited  in  area  because  of  climatic  and  soil  conditions.  ‘Where 
the  areas  of  two  crops,  that  hold  the  same  place  in  a rotation 
and  require  labor  at  the  same  time  of  year,  overlap,  the  crop 
with  the  more  limited  area  will  be  the  strongest  competitor  for 
the  use  of  the  land  especially  suited  to  its  production.  And 
the  tendency  is  for  the  crop  with  the  wider  area  to  be  grown 
on  land  not  required  for  the  crop  with  the  limited  area. 

This  point  may  be  made  clear  by  reference  to  a concrete  ex- 
ample. A piece  of  land  in  France  said  to  be  as  fine  land  as  exists 
in  the  world  for  wheat  production,  is  suited  also  to  the  pro- 
duction of  grapes  that  make  a brand  of  wine  very  highly  prized. 
The  areas  suited  to  the  production  of  this  brand  of  wine  are 
very  limited,  but  the  areas  suited  to  wheat  production  are 
abundant.  To  exclude  wheat  from  the  wine  land  affects  the 
supply  of  wheat  but  little,  but  to  exclude  the  wine  from  any  ap- 
preciable portion  of  the  limited  area  suited  to  its  culture  for 
the  manufacture  of  this  special  brand  of  wine  would  materially 
reduce  the  supply  and  result  in  a rising  price,  which  would  give 
the  vine  a greater  power  in  competing  for  the  use  of  the  land. 

It  happens  that  while  corn  has  more  extended  uses  and  more 
exclusive  uses  than  sugar  beets,  the  world  has  a much  greater 
area  physically  suited  to  beet  culture  than  to  corn  culture.  It 
is  hardly  probable,  therefore,  that  the  sugar  beet  will  ever  be 
able  to  compete  with  corn  on  even  terms  in  the  corn  belt  of  the 
United  States. 

Wages  and  interest  vary  directly  with  tlqe  opportunity  for 
the  profitable  employment  of  labor  and  capital  and  inversely 
with  the  supply  of  these  factors.  This  is  another  economic  law 
which  has  received  too  little  attention  in  the  promotion  of  the 
sugar  beet  industry  in  the  United  States  where  opportunities 
for  productive  labor  are  abundant,  and  labor  and  capital  scarce 
in  comparison  with  our  European  competitors. 


Economics  in  Agricultural  Education  and  Research.  97 


Other  conditions  being  equally  favorable,  a country  in  which 
laborers  and  equipments  are  relatively  scarce  cannot  success- 
fully compete  in  the  production  of  crops  requiring  relatively 
large  applications  of  labor  and  capital,  unless  the  product  be  a 
perishable  one  which  will  not  stand  long  shipments,  or  one  with 
a very  low  specific  value  on  which  the  freight  would  be  very  high 
per  dollar’s  worth  of  product.  High  wages  put  the  sugar  beet 
industry  at  a disadvantage  in  the  United  States  and  this  fact 
points  to  the  wisdom  of  our  producing  the  other  crops  which 
require  less  labor  than  beets  or  in  the  growing  of  which  Euro- 
pean labor  is  not  generally  in  competition. 

While  the  beet  sugar  industry  lends  itself  well  to  illustrating 
the  necessity  of  studying  economic  forces  as  well  as  the  physical 
and  biological  forces  with  which  the  farmer  has  to  deal  in  order 
that  a practical  conclusion  may  be  drawn,  this  necessity  exists  in 
every  line  of  production.  Tobacco,  alfalfa,  wheat,  barley,  oats, 
corn,  cotton,  potatoes,  beans,  grapes,  apples,  peaches,  oranges, 
lemons,  dairy  products,  beef,  pork,  mutton,  wool,  and  all  other 
agricultural  products  have  to  be  produced  where  the  physical 
environment  is  suitable,  but  within  these  limits,  conditions  with 
repect  to  labor,  capital,  markets,  relative  profitableness  of  com- 
peting crops  or  live  stock  become  prime  factors  in  determining 
what  to  produce  in  a given  place.  Silk  and  tea  can  be  pro- 
duced in  the  United  States,  but  on  account  of  the  difference  in 
labor  conditions  here  and  in  the  competing  countries  of  the 
Orient,  these  products  can  be  imported  more  cheaply  than  they 
can  be  produced  at  home. 

The  Problem  of  Intensity  of  Culture 

The  question  of  the  proper  degree  of  intensity  of  cul- 
ture is  the  problem  of  the  proper  proportions  between  land, 
labor,  and  capital  in  the  cultivation  of  a given  crop.  The  prob- 
lem of  proper  proportions  extends  to  every  detail  of  the  busi- 
ness of  farming.  For  example,  the  proportion  between  the  ex- 
penditure for  feed  and  for  shelter  for  a milk  cow  is  a problem 
similar  to  that  of  the  proper  degree  of  intensity  of  culture. 
Proper  proportions  are  determined  in  part  by  physical  condi- 
tions, in  part  by  biological  conditions ; and  in  part  by  economic 
considerations.  In  all  cases  the  relative  profitableness  of  the 
different  methods  is  the  determining  factor.  Cheap  land  and 


98  Wisconsin  Experiment  Station. 

expensive  labor  favor  extensive  culture.  Cheap  feed  and  expen- 
sive building  materials  favor  the  use  of  more  feed  and  less 
shelter. 

The  Tenant  Problem 

The  problem  of  the  proper  adjustment  of  the  relations  be- 
tween landlord  and  tenant  has  often  been  regarded  as  belonging 
to  the  economist,  because  the  human  relations  dominate,  and  yet 
in  the  adjustment  of  the  relations  between  landlords  and  tenants 
the  specialists  in  the  other  sciences  must  be  called  upon.  The 
custom  of  binding  the  tenant  to  a particular  mode  of  farming 
with  a view  of  avoiding  soil  exhaustion  makes  a demand  for 
knowledge  which  will  harmonize  these  requirements  wTith  phy- 
sical and  biological  laws  as  well  as  with  economic  laws. 

A most  striking  example  of  the  way  in  which  certain  biological 
and  chemical  knowledge  is  essential  to  right  adjustment  of  the 
complex  interests  of  landlord  and  tenant,  is  the  work  done  on 
the  experiment  plots  at  Rothamsted  in  the  solution  of  the  tenant 
problem  in  England.  Centuries  ago  a wise  man  conceived  the 
idea  that  if  tenant  farmers  holding  from  year  to  year  were  paid, 
at  the  termination  of  their  tenancies,  for  the  fertilizer,  the  til- 
lage, etc.,  which  they  had  put  into  or  upon  the  land  and  from 
which  they  had  not  yet  realized  the  benefits,  this  would  induce 
them  to  use  fertilizer  as  freely  and  to  till  the  soil  as  thoroughly 
as  if  they  owned  the  land.  The  idea  was  a good  one,  but  gen- 
eration after  generation  went  by  without  much  success  in  its  ap- 
plication, because  no  one  knew  how  much  of  the  fertilizer  was 
used  by  the  first  crop  and  how  much  by  the  second,  etc.  No  one 
knew  what  variation  there  was  on  different  soils,  with  different 
crops  and  with  different  fertilizers,  in  the  extent  to  which  the 
fertilizer  would  be  realized  upon  the  first  year.  The  scientists 
of  Rothamsted  collected  data  on  this  point,  and  when  the  work 
of  the  rural  economist  was  supplemented  by  the  work  of  the 
chemist,  the  soil  physicist  and  the  plant  physiologist,  the  system 
of  compensation  for  unexhausted  improvements  became  the 
essential  feature  of  the  English  system  of  adjusting  the  relation 
between  landlord  and  tenant. 


Economics  in  Agricultural  Education  and  Research.  99 


The  Changeable  Character  of  Economic  Conditions 

Thus  far,  the  aim  has  been  to  show  by  a few  concrete  examples 
the  place  of  economics  as  one  of  the  fundamental  sciences  which 
must  be  used  in  making  a complete  study  of  any  problem  which 
confronts  the  farmer.  In  one  respect  the  economic  phase  of  the 
farm  problem  is  somewhat  different  from  the  physical  and  bio- 
logical phases. 

The  human  factors  are  ever  changing.  The  conditions  with 
respect  to  the  labor  supply,  the  supply  of  capital,  the  quality  of 
the  labor  and  the  usefulness  of  the  equipments,  the  facilities 
for  marketing,  the  prices  of  the  products,  etc , are  changing 
constantly.  For  this  reason  the  conclusion  which  is  correct  at 
one  time  in  a given  locality,  as  to  what  crops  to  grow  and  with 
what  degree  of  intensity  they  should  be  cultivated,  ceases  to  be 
correct  at  another  time.  Economic  questions  relating  specifically 
to  “what  to  do”  and  “how  to  do  it”  never  are  and  never  can 
be  settled  once  for  all.  Only  temporary  and  local  solutions  are 
possible. 

The  economic  problem  becomes  one  of  continuous  readjust- 
ment of  the  farmer’s  activities,  to  changes  in  the  human  environ- 
ment. Let  it  be  made  clear,  however,  that  it  is  conditions  of 
life  which  change,  not  the  laws  in  accordance  with  which 
economic  forces  operate.  Economic  forces  act  as  consistently  as 
physical  laws.  This  truth  is  often  obscured  by  the  fact  that 
economic  forces  are  so  numerous  and  operate  in  so  many  direc- 
tions and  with  such  variation  in  intensity  that  it  is  exceedingly 
difficult  to  hold  in  mind  all  of  the  forces  which  are  operating  at 
a given  time  and  place,  and  draw  a correct  conclusion  as  to  the 
probable  result. 

The  first  work  of  the  economist  is  to  elucidate  the  economic 
forces  which  should  be  considered  in  deciding  “what  to  do” 
and  “how  to  do  it.”  So  far  as  possible  he  should  measure  these 
forces,  and  assist  in  drawing  practical  conclusions,  With  these 
economic  forces  which  determine  profits  clearly  in  mind,  the 
farmer,  assisted  by  the  station  economist  and  accountant,  should 
be  able  to  organize  his  farm  so  that  it  will  yield  maximum  re- 
sults under  given  conditions  and  to  adjust  his  farming  from 
time  to  time  in  such  ways  as  may  be  made  necessary  by  changes 


100  Wisconsin  Experiment  Station. 

in  the  prices  of  land,  labor,  and  equipment,  and  the  prices  of  the 
various  products  of  the  farm. 

This  work  demands  men  who  are  students  of  economic  forces 
and  who  are  thoroughly  familiar  with  the  agricultural  indus- 
try. Every  farmer  knows  something  of  economic  forces  because 
he  deals  with  them  from  day  to  day.  This  is  likewise  true  re- 
garding the  physical  and  biological  forces.  All  we  can  hope  to 
do  is  to  help  him  to  a more  perfect  knowledge. 

Necessity  of  Cooperation  with  Specialists  in  Other 

Sciences 

The  work  of  the  specialists  in  agricultural  economics  must  be 
carried  on  in  close  cooperation  with  the  specialists  in  the  physical 
and  the  biological  sciences.  The  business  of  the  agricultural 
experiment  station  is  to  help  farmers  solve  the  problems  which 
confront  them.  These  problems  are  broader  than  any  one 
branch  of  science.  The  director  of  the  experiment  station  stands 
in  a position  to  select  specialists  in  each  of  the  fundamental  sci- 
ences which  contribute  to  the  discovery  of  the  hidden  forces 
with  which  the  farmer  has  to  deal  and  to  organize  these  special- 
ists into  one  unified  scheme  for  solving  problems. 

The  success  of  agricultural  economics  in  the  station  depends 
largely  upon  this  close  coordination  of  the  whole  group  of  spe- 
cialists. The  economic  problems  are  all-comprehending  but  eco- 
nomics is  not  all-sufficient.  The  economist  has  either  to  know  or 
to  assume  the  results  of  the  specialists  in  the  physical  and  bio- 
logical sciences.  If  he  has  to  depend  upon  assumptions,  his 
conclusions  will  be  speculative  and  sterile.  The  only  practical 
and  vitalizing  way  is  for  the  men  conversant  with  the  different 
groups  of  forces  to  cooperate  in  carrying  on  the  investigations 
essential  to  a well  balanced  answer  to  the  questions  raised  by  the 
farmers. 


Economics  in  Agricultural  Education  and  Research.  101 


THE  SCOPE  OF  AGRICULTURAL  ECONOMICS 

For  convenience  in  teaching,  agricultural  economics  may  be 
divided  into  two  parts.  The  first  part  deals  with  the  laws  of  pro- 
duction, the  second  part  treats  of  the  laws  of  value.  In  the  first 
part,  the  object  is  to  study  the  economic  forces  and  conditions 
and  formulate  the  principles  in  harmony  with  which  the  farmer 
must  organize  and  operate  his  farm  in  order  to  secure  maximum 
results  in  production.  In  the  second  part,  the  aim  is  to  study 
the  forces  and  conditions  which  determine  the  prices  of  farm 
products,  the  prices- paid  for  services  necessary  to  the  production 
and  marketing  of  products,  and  the  prices  paid  for  the  use  of 
land  and  equipments. 

In  the  economics  of  production,  the  problem  is  primarily  that 
of  taking  the  shortest  road  to  the  desired  end,  the  economy  of 
energy  in  satisfying  human  wants.  In  the  study  of  values  the 
problem,  first  of  all,  is  to  know  why  prices  are  what  they  are 
and  what  they,  are  tending  to  be  in  the  future,  but  includes,  also, 
the  whole  problem  of  justice  in  distribution.  Justice  in  distri- 
bution requires  that  each  person  who  has  contributed  to  the 
bringing  of  the  product  into  existence,  or  to  the  putting  of  it  in 
the  right  place  at  the  right  time  or  its  conversion  into  the  right 
form  for  the  consumer,  should  receive  a fair  compensation  for 
his  services. 

The  two  parts  are  closely  related.  The  situation  with  regard 
to  values  forms  the  background  on  which  the  principles  of  pro- 
duction must  be  worked  out.  Relative  prices  and  relative  costs 
determine  what  the  farmer  can  afford  to  produce.  The  relation 
between  the  value  of  labor,  the  value  of  land,  and  the  value  of 
machinery  and  other  equipments  determines  the  proportions  in 
which  these  factors  should  be  made  use  of.  It  determines  the 
proper  degree  of  intensity  of  culture.  In  the  application  of 
economic  principles  to  any  specific  problem,  the  laws  of  produc- 
tion and  the  laws  of  value  must  he  held  in  mind  and  applied 
simultaneously.  But  when  the  student  is  becoming  familiar 
with  economic  forces  it  is  better  first  to  trace  the  operation  of 
the  economic  laws  under  given  conditions  with  respect  to  values, 
then  study  carefully  the  effect  of  a change  in  the  value  of  the 
products,  a change  in  the  relative  value  of  the  products,  a change 


102 


Wisconsin  Experiment  Station. 


in  the  cost  of  the  factors  of  production,  and  a change  in  the  rela- 
tive costs.  The  problems  of  crop  selection  and  of  intensity  of 
culture  stand  out  prominently  as  demanding  a treatment  of  the 
laws  of  production  under  conditions  of  assumed  values.  The 
laws  of  production  having  been  mastered  the  student  should 
study  the  causes  of  values  and  of  changes  in  values. 

The  Economic  Principles  of  Production  - 

Factors  of  Production  and  Their  Characteristics.  The  proper 
1 beginning  in  the  study  of  the  economics  of  agricultural  produc- 
tion is  a study  of  the  economic  characteristics  of  the  factors  of 
production;  land,  labor  and  equipment.  The  variability  in  the 
usefulness  of  the  different  units  of  each  of  these  factors,  the 
relative  abundance  of  the  supply,  the  rate  of  increase  in  the  sup- 
ply of  each  factor,  and  the  quality  of  the  new  increments  of  sup- 
ply are  all  matters  which  should  be  clearly  before  the  minds  of 
students  before  any  attempt  is  made  to  study  the  correlation 
of  these  factors  in  a system  of  agricultural  production. 

Correlation  of  Factors  of  Production.  Wdien  the  economic 
characteristics  of  the  factors  of  production  are  clear  in  the  mind 
of  the  student  he  may  be  introduced  to  the  economic  principles 
which  underlie  the  correlation  of  the  factors  in  a system  of  agri- 
cultural production. 

The  first  problem  of  correlation  pertains  to  the  grades  of 
farmers,  laborers,  land,  and  equipments  which  should  be  associ- 
ated. Should  the  most  efficient  farmer  occupy  the  most  produc- 
tive land  and  employ  the  most  efficient  laborers  and  equipments  ? 
flow  may  the  farm  operator  take  advantage  of  differences  in 
the  capacity  of  laborers  in  planning  the  work  of  the  farm,  etc.  ? 

The  second  problem  of  correlation  pertains  to  the  proportion 
in  which  land,  labor  and  equipments  should  be  combined  on  a 
given  farm  in  a given  line  of  production.  This  is  the  problem 
of  intensity  of  culture  extended  to  every  activity  of  the  farm. 

Choice  of  Lines  of  Production.  The  problem  of  what  to 
produce  on  a given  farm  is  a complex  one  because  physical  con- 
ditions usually  make  farming  a diversified  industry.  The  prob- 
lem becomes  that  of  determining  upon  the  several  lines  of  pro- 
duction that  will  complement  each  other  in  their  demands  for 
man  and  horse  labor,  and  will  give  the  most  profitable  employ- 


■try  was  concentrated  in  Vermont,  Western  Massach  usetts,  and  New  York,  with  important  begin- 
k nings  of  the  industry  in  western  Pennsylvania  and  Ohio. 


Economics  in  Agricultural  Education  and  Research.  103 


ment  for  the  seasons  of  the  year  in  which  they  demand  the 
farmer’s  time.  This  problem  is  all  the  more  difficult  for  the  rea- 
son that  the  crops  may  be  competitive  during  seasons  of  cultiva- 
tion and  complementary  at  other  times. 

The  Size  of  the  Farm.  The  amount  of  business  which  can  be 
brought  under  one  management  to  best  advantage  is  the  problem 
here  involved.  Various  measures  of  the  size  of  the  farm  business 
unit  have  been  used.  For  example,  the  number  of  acres,  the 
number  of  men  employed,  the  number  of  horses,  the  number  of 
plows,  the  number  of  cattle  or  sheep  and  the  gross  product  in 
value  are  measures  which  may  be  applied.  The  proper  size  varies 
with  the  character  of  the  land,  the  character  of  the  productions 
and  the  character  of  the  farmer  and  his  family.  Hence  no  one 
size  of  farm  is  best  for  all  times  and  places.  To  develop  the 
principles  to  be  followed  in  determining  the  proper  size  of  a 
farm,  is  the  work  of  the  economist. 

Economic  Status  of  the  Farmer.  The  amount  of  wealth  pos- 
sessed by  the  farmer  is  an  important  factor  in  determining  the 
extent  to  which  he  is  able  to  gain  control  of  the  land,  labor  and 
equipment  essential  to  the  organization  of  his  business  in  an 
economical  manner.  Land  tenure,  labor  systems,  and  credit  in- 
stitutions should  be  studied  from  the  standpoint  of  facilitating 
the  economical  organization  of  the  farm. 

The  State<  in  its  Relation  to  Agricultural  Production.  The 
study  of  agricultural  production  should  not  be  closed  without 
viewing  the  subject  from  the  standpoint  of  the  common- 
wealth. What  kind  of  farming  is  best  in  the  long  run  for  the 
people  of  a state,  of  a nation,  of  the  world?  The  soil  fertility, 
the  education  of  the  farmer,  the  improvement  of  the  breeds  of 
plants  and  animals,  are  questions  which  should  be  carefully  con- 
sidered from  the  standpoint  of  the  possibilities  of  state  activity. 
State  activities  in  promoting  agriculture  should  take  into  account 
economic  as  well  as  physical  and  biological  forces.  The  agrarian 
statesman,  as  well  as  the  farmer,  to  be  successful,  must  act  in 
harmony  with  economic  laws. 


104 


Wisconsin  Experiment  Station. 


Forces  and  Conditions  Which  Determine  Value 

When  this  phase  of  the  subject  has  been  reached,  the  aim  is 
to  study  all  the  forces  and  conditions  which  affect  the  values  of 
the  products  of  the  farm  and  of  the  factors  of  production ; land, 
labor,  and  equipment.  This  study  should  be  prefaced  by  a gen- 
eral analysis  of  the  forces  which  determine  values,  the  laws  of 
demand  and  the  conditions  of  supply,  whether  they  be  competi- 
tive, customary,  or  monopolistic. 

Prices  and  the  Marketing  of  Farm  Products.  The  applica- 
tion of  the  theory  of  value  in  determining  the  causes  of 
high  or  low  prices  involves  a study  of  money,  credit,  trans- 
portation systems,  the  organized  systems  of  marketing,  etc. 
These  institutions  play  an  important  part  in  giving  an  outlet 
for  the  productions  of  the  farm.  The  services  of  all  these  insti- 
tutions must  be  remunerated  out  of  the  price  paid  by  the  con- 
sumer for  a given  product.  The  farmer  is  interested  in  knowing 
of  these  institutions  first  of  all  that  he  may  make  the  best  use  of 
them,  and  he  is  highly  interested  in  the  justice  of  the  charges 
made  for  their  services,  for  this  affects  the  return  which  he  can 
secure  for  his  products. 

The  general  study  of  the  forces,  conditions,  and  institutions 
which  affect  prices  of  products  should  be  followed  by  concrete 
studies  of  the  forces  and  conditions  affecting  the  price  of  indi- 
vidual products.  The  price  of  wheat  should  be  studied  from  all 
standpoints.  The  conditions  of  production  throughout  the  world, 
the  world’s  demand  for  wheat,  the  facilities  for  marketing  wheat, 
the  fairness  of  the  charges  made  by  middlemen  who  handle 
wheat,  etc.,  should  be  studied  in  a concrete  way,  not  simply  rea- 
soned about,  but  the  facts  sought  out  and  measured.  This 
method  should  be  applied,  with  proper  modification  to  fit  the 
case,  to  each  of  the  cereals,  each  of  the  vegetables,  each  of  the 
fruits,  to  cotton,  tobacco,  wool,  mutton,  pork,  beef,  milk,  cheese, 
butter,  eggs,  and  to  every  other  product  of  the  farm.  Nothing  else 
in  agricultural  economics  is  of  more  importance  than  this  thor- 
ough grounding  in  the  forces  and  conditions  which  affect  the 
prices  of  each  of  the  products  which  the  farmer  is  producing. 
Prices  of  products  determine  what  is  proper  management  of  a 
farm.  The  farmer  must  keep  his  eyes  upon  the  market  as  well 
as  upon  the  farm,  if  he  would  guide  his  energy  into  the  lines 


Economics  in  Agricultural  Education  and  Research.  105 

of  maximum  profits.  (See  bulletin  209  of  the  Wisconsin  Experi- 
ment Station.) 

Prices  and  the  Purchasing  of  Goods  and  Services.  The  study 
of  values  should  be  prosecuted  with  equal  vigor  in  the 
fields  of  the  farmer’s  purchases.  The  prices  of  family  supplies 
, and  of  tools  and  machinery  should  be  studied.  The  price  of  labor 
should  be  considered  both  from  the  standpoint  of  the  cost  to  the 
farmer  and  from  the  standpoint  of  the  opportunity  of  the  laborer 
to  improve  his  economic  status.  The  rent  of  land,  the  farmer’s 
profits,  the  rate  of  return  on  capital,  and  the  market  price  of 
land,  are  questions  deserving  the  most  thorough  study.  The 
farmer  may  do-all  else  well,  but  if  he  pays  too  much  for  his  land 
i his  efforts  will  prove  fruitless. 

Prices  and  the  Status  of  the  Farmer.  The  workings  of  the 
j.  forces  which  determine  prices  in  all  these  lines  determine, 
in  a large  measure,  whether  the  succeeding  generations 
of  American  farmers  are  to  lose  or  gain  in  their  hold  upon 
the  bases  of  production.  The  operation  of  the  value-deter- 
mining forces  is  of  prime  importance  in  determining  whether 
landownership  or  tenancy  shall  be  the  most  common  method  of 
holding  land,  and  in  determining  the  kind  and  the  quality  of 
food,  clothing,  shelter,  education,  and  other  bases  of  human- 
life  which  the  farmer  may  command. 

r The  State  s Fetation  to  the  Quality  and  Price  of  Goods  and 
f Services.  Due  attention  should  be  given  to  activities  of  the  gov- 
ernment m their  actual  and  possible  relations  to  the  problems  of 
buying  and  marketing,  whether  it  be  in  the  inspection  of  the 
products  sold  by  the  farmers  or  the  inspection  of  the  charges 
made  for  their  services  by  monopolistic  producers  or  middlemen. 

On  the  other  hand  the  charges  made  by  the  government  for 
its  services,  whether  these  charges  be  in  the  nature  of  direct  or 
indirect  taxes,  should  be  viewed  from  the  standpoint  of  their 
fairness  and  their  influence  upon  the  activities  and  the  status  of 
the  farmer.  (See  appendix  for  chapter  headings  of  a course  in 
Agricultural  Economics.) 


106 


Wisconsin  Experiment  Station. 


) 


METHODS  USED  IN  THE  STUDY  OF  ECONOMIC 
PROBLEMS  IN  AGRICULTURE 

Our  knowledge  of  the  character  of  economic  forces  comes 
through  reasoning  based  upon  the  available  facts.  In  economics  j 
as  in  other  sciences,  the  work  of  the  student  consists  in  gathering 
facts,  sifting  and  classifying  them,  formulating  hypotheses,  gath- 
ering more  evidence  with  which  to  test  the  tentative  conclusions, 
until  all  the  relevant  facts  have  been  considered  and  the  correct  ; 
conclusions  drawn.  Through  these  processes  it  should  be  pos- 
sible in  time  to  approximate  the  truth  regarding  the  operation  of  : 
particular  economic  forces.  But  the  economist  is  never  certain 
of  having  considered  all  of  the  facts,  and  he  is  ever  hoping  to  j 
formulate  a new  hypothesis  which  will  more  completely  explain 
the  evidence  in  his  possession. 

Theoretical  work  in  general  may  he  divided  into  two  classes,  j 
the  sterile  and  the  fruitful.  The  sterility  of  the  theories  of  the 
one  class  is  usually  due  to  failure  to  see  the  problem  m its  entire 
setting.  The  conclusions  are  sterile,  as  a rule  not  because  of 
illogical  thinking,  but  because  the  premises  are  incomplete  and 
hence  the  conclusions  are  erroneous.  The  fruitfulness  of  the 
other  class  of  theories  is  due  to  the  fact  that  they  are  m harmony 
with  the  facts  and  become  a guide  to  the  mind  in  comprehending  j 
the  direction  and  strength  of  the  forces  with  which  the  practical  i 
man  has  to  deal  and  hence  increase  the  accuracy  of  his  judg- 
ments regarding  the  probable  conditions  of  the  future  with  which 
he  has  to  make  his  present  actions  harmonize. 

The  danger  of  holding  to  half  truths  is  very  great  in  the  field 
of  economics.  The  forces  involved  are  so  numerous  and  the  facts  | 
so  scattered  that  even  the  most  careful  student  is  in  danger  of 
placing  too  great  reliance  upon  a premature  conclusion.  And 
yet  practical  men  everywhere  are  constantly  dealing  with  eco- 
nomic forces.  Day  after  day  they  are  passing  judgment  on  the 
future  action  of  these  forces.  Practical  business  men  often  show 
a clearer  grasp  of  the  operation  of  economic  forces  than  do  the 
economists  of  the  chair.  This  is  because  the  business  men  are 

dealing  directly  with  these  forces.  . 

The  student  must  study  economic  forces  in  operation  it  he 
would  understand  their  character.  The  world  of  economic  activi- 


Economics  in  Agricultural  Education  and  Research.  107 

ties  should  be  the  laboratory  of  the  economist  and  the  records  of 
these  activities  should  constitute  his  library.  The  success  of  the 
student  depends  equally  upon  his  ability  to  gather  data  and  Uls 
ability  to  draw  correct  mterences.  It  may  be  true  in  some  sub- 
jects that  a person  who  is  not  capable  of  drawing  conclusions  will 
be  able  to  work  independently  and  contribute  to  the  subject  by 
gatheiing  data  which  others  may  use,  but  m the  field  of  economics 
the  pioblems  are  so  complex  that  in  order  to  secure  valuable  re- 
sults the  two  processes  must  be  employed  simultaneously.  No 
student  should  undertake  independent  research  work  in  econom- 
ics who  is  not  a good  iogician.  He  must  be  capable  of  correct 
reasoning.  He  must  be  capable  of  drawing  the  right  inference 
from  given  facts  and  of  remembering  the  limitations  of  the  basis 
of  his  reasoning.  When  a working  hypothesis  is  formulated,  it 
should  be  looked  upon  as  a means  to  an  end,  not  as  an  end  in  it- 
self. In  other  words  he  must  be  capable  of  independent  work  in 
the  field  of  economic  theory.  Possessing  this  qualification,  the 
student  should  devote  most  of  his  time  to  the  gathering  of  data 
which  may  form  the  basis  of  generalizations  regarding  the  char- 
acter and  operation  of  economic  forces. 

It  is  not  the  purpose  of  this  paper  to  elaborate  in  a systematic 
way  the  whole  subject  of  deductive  and  inductive  reasoning,  but 
to  outline  some  of  the  methods  which  have  proved  useful  in 
gathering  facts  and  putting  them  in  such  form  that  the  mind 
can  comprehend  them  in  their  relation  to  each  other.  When 
this  work  is  well  done  the  drawing  of  conclusions  requires  little 
time. 

The  methods  which  will  be  discussed  here  may  be  classified  as  : 
Historical,  Geographical,  Statistical,  Accounting,  and  Experi- 
mental. These  methods  are  not  exclusive.  The  student  interprets 
history  in  terms  of  geography.  Much  of  his  best  data  for  his- 
torical and  geographical  study  are  prepared  by  the  statistician 
and  he  resorts  to  the  accounting  and  experimental  methods  for 
a more  detailed  study  of  the  present  condition  at  a given  place, 
and  to  explain  geographical  differences.  The  historical  and  geo- 
graphical methods  give  breadth  of  view  and  a basis  for  sound 
judgment  as  to  the  trend  of  affairs.  The  accounting  and  experi- 
mental methods  give  depth  of  insight  and  the  basis  of  keen  anal- 
ysis. 


108  Wisconsin  Experiment  Station. 

« 

The  Historical  Method 

In  the  study  of  economic  forces,  much  is  gained  by  tracing 
their  operations  through  a considerable  period  of  time.  Eco- 
nomic forces  are  not  easily  measured,  and  they  are  so  numerous, 
of  such  varying  strength,  and  so  often  operate  in  opposite  direc- 
tions that  at  any  given  moment  it  is  difficult  to  make  an  estimate 
of  the  future  resultant  of  these  forces,  unless  the  changes 
wrought  by  them  in  the  past  can  be  resorted  to  as  a basis  of 
judgment. 

The  federal  census  for  1900  showed  that  more  than  a third  of 
the  farmers  of  the  United  States  were  tenants  and  about  a third 
of  the  farms  operated  by  owners  were  mortgaged.  Without  re- 
sorting to  the  historical  method  it  would  have  been  impossible 
to  know  whether  the  forces  making  for  tenancy  were  overbalan- 
cing the  forces  making  for  ownership,  or  vice  versa.  It  might 
be  inferred  by  one  viewing  these  facts  without  this  historical 
setting  that  ownership  had  once  been  universal  and  that  the 
owners  had  lost  money,  mortgaged  their  farms,  lost  their  titles 
to  the  land  and  become  tenant  farmers.  On  the  other  hand,  one 
might  infer  that  farmers  were  using  tenancy  and  the  mortgage 
as  means  of  making  transition  from  landless  laborers  to  the  free 
ownership  of  land.  This  illustration  is  to  the  point  because  simi- 
lar inferences  were  made  in  1880  when  statistics  of  land  tenure 
were  collected  for  the  first  time. 

At  the  present  time,  with  the  changes  of  thirty  years  recorded 
at  ten  year  intervals,  it  is  possible  to  demonstrate  clearly  the 
trend  of  affairs  during  that  period,  and  to  describe  many  of  the 
forces  which  have  been  operating.  The  available  materials  show 
that  young  men  do  very  generally  rise  through  the  successive 
stages  of  tenant  farmers  and  mortgaged  owners  to  the  free  own- 
ership of  farms,  but  the  data  show  also  that  there  has  been  a 
retardation  in  this  movement  and  that  longer  time  is  required 
to  make  this  movement  recently  than  in  earlier  years.  The 
census  data  for  1890  and  for  1900  show  that  older  farmers  are 
generally  owners,  while  tenancy  is  most  common  among  young 
farmers.  By  comparing  the  data  for  the  two  periods,  it  be- 
comes clear  that  some  force  is  retarding  the  movement  from 
tenancy  to  ownership,  for  a smaller  percentage  of  those  of  the 
various  ages  were  owners,  and  a larger  percentage  were  tenants 
in  1900  than  in  1890.  This  is  illustrated  in  Figure  1.  Illinois 


Economics  in  Agricultural  Education  and  Research.  109 


o 


110 


Wisconsin  Experiment  Station. 


is  used  for  this  illustration  because  tenancy  is  more  common 
m tnat  state  than  in  any  other  part  of  the  North.  The  illus- 
tration shows  that  tlie  percentage  of  owners  among  young  farm- 
ers is  very  small,  but  that  ownership  increases  with  the  age 
of  a farmer  and  that,  of  the  farmers  55  years  of  age  and  over, 
about  85  per  cent  are  owners.  By  comparing  the  situation  in 
1890  and  1900  for  each  age  group,  it  becomes  clear  that  while 
there  is  a movement  toward  land  ownership,  as  the  farmers 
grow  older,  it  is  true,  however,  that  a smaller  proportion  of  the 
tanners  of  eacn  age  group  were  owners  in  1900  than  in  1890, 
snowing  retardation  in  the  movement  from  tenancy  to  free 
ownership.  The  right  hand  illustration  in  Figure  1 shows  the 
reverse  situation  with  regard  to  tenancy,  that  tenancy  is  most 
common  in  the  younger  age  groups  and  gradually  declines. 
A careful  study  of  the  facts  now  available  shows  that  many 
forces  are  in  operation,  some  making  for  dependent  tenants 
others  for  independent  landowning  farmers. 

Another  example  of  the  historical  study  of  economic  forces  is 
afforded  by  the  sheep  industry  in  the  United  States.  Between 
1840  and  1850  there  wras  a decline  in  the  number  of  sheep  kept  in 
parts  of  Vermont  and  in  the  eastern  part  of  New  York,  but  the 
marked  change  was  in  Ohio  and  Michigan,  where  there  was  a 
great  increase.  (Figures  3 and  4.)  The  tendencies  were  the 
same  in  Vermont,  New  York,  Ohio  and  Michigan,  between 
1850  and  1860,  (Figure  5),  with  an  important  beginning  of  the 
sheep  industry  in  Texas,  California  and  Oregon.  The 
statistics  for  New  Mexico  for  1850  do  not  give  a 
measure  of  any  change  which  may  have  taken  place  since  1840, 
but  simply  show  the  status  in  1850.  New  Mexico  was  not  in 
the  United  States  in  1840,  hence  there  was  no  census  taken. 
But  the  Navajo  Indians  were  engaged  in  sheep  growing  there,  so 
the  grazing  of  sheep  may  be  a very  old  industry  in  New  Mexico. 

The  decade  from  1860  to  1870  brought  a reaction  in  north- 
eastern Ohio  and  the  beginnings  of  the  concentration  of  the 
sheep  industry  of  Texas  in  the  dry  lands  of  the  South.  (Figure 
6.)  Both  of  these  movements  continued  during  the  next  decade. 
Figure  7.)  By  1880,  Vermont  had  almost  ceased  to  be 
a sheep  state,  and  the  sheep  of  New  York  were  but  a 
handful  in  comparison  to  the  number  in  1840,  but  the  begin- 
nings of  the  new  industry  in  the  Rocky  Mountain  states  were 
already  important  in  Colorado,  Wyoming,  and  Montana.  Dur- 


Economics  in  Agricultural  Education  and  Research.  Ill 

ing  tlie  next  two  decades  the  development  of  the  sheep  indus- 
try in  the  mountain  states  continued,  but  between  1890  and 
1900  a marked  decline  is  shown  in  California,  Texas,  Wiscon- 
sin, Michigan,  and  Ohio.  (Figures  8 and  9.) 

When  the  maps  for  1810  and  1900  are  compared,  it  becomes 
evident  that  during  the  sixty  years  the  sheep  industry  made  a 
complete  shift  with  the  exception  of  a few  counties  in  Pennsyl- 
vania and  Ohio,  so  far  as  the  concentrated  centers  of  production 
are  concerned.  By  reading  the  agricultural  papers  of  this  pe- 
riod one  finds  many  references  to  these  changes.  A well  known 
Merino  breeder  of  Vermont  in  one  decade  is  heard  from  in  the 
next  decade  from  California  where  he  is  in  the  same  business. 
The  method  of  driving  sheep  from  Ohio  to  Texas  is  described 
and  in  fact  the  student  who  has  the  time  and  ability,  can  easily 
reconstruct  the  whole  movement  of  the  sheep  industry  from  the 
days  when  Vermont  and  New  York  led  in  this  industry  to  the 
present  day  with  the  Rocky  Mountain  states  in  the  dominant 
position.  (See  Figures  3-9.) 

The  story  is  only  half  told,  however,  until  it  is  explained.  To 
explain  these  changes  it  becomes  necessary  to  write  the  history 
of  the  expansion  of  American  agriculture.  The  competition  of 
the  various  farm  enterprises  has  played  an  important  part.  The 
result  of  this  competition  is  determined  by  geographic  factors. 
Topography,  climate,  and  nearness  to  the  market  are  important 
examples. 

In  Vermont,  New  York,  northeastern  Ohio  and  in  Wis- 
consin, wool  growing  was  driven  out  by  the  dairy  industry. 
This  is  indicated  by  contemporaneous  literature.  Statistics  of 
the  dairy  industry  were  not  collected  in  1840,  but  the  milk 
production,  as  shown  for  1900,  in  Figure  2,  indicates  the  pres- 
ence of  this  industry  at  the  latter  date  in  the  regions  which  were 
in  the  earlier  decades  important  centers  of  the  sheep  industry. 
The  economic  principle  involved  in  simple.  Wool  is  durable, 
dairy  products  are  perishable.  The  value  of  wool  per  pound  is 
much  greater  than  milk  and  has  often  been  greater  than  butter 
and  cheese.  The  dairyman  at  a distance  from  the  market  for 
dairy  products  cannot  compete  with  the  one  near  the  market  so 
well  as  the  shepherd  in  the  distant  hills  and  downs  can  compete 
with  the  wool  grower  near  the  center  of  population. 

Beef  cattle  replaced  sheep  in  Texas,  the  development  of  agri- 
culture and  fruit  growing  by  irrigation  was  an  important  factor 


112 


Wisconsin  Experiment  Station. 


6 

a 

o 

c 


Z a 
c “ 


5 K 
— « 

Co 
% ^ 

C “H 


^ ej 
c S. 


~ c 
c a 
o 


« a 
- o 


.2 

EH 


P 

to 

£ 


Economics  in  Agricultural  Education  and  Research.  113 

in  California,  and  tariff  legislation  played  an  important  part  in 
forcing  out  the  sheep  in  Ohio  and  Michigan  during  the  nineties. 
A careful  study  of  the  operation  of  competitive  forces,  with  and 
without  artificial  price  levels,  enables  the  student  to  discover 
the  workings  of  the  economic  laws  which  have  wrought  the 
changes. 

The  Geographical  Method 

A study  of  the  types  of  farming  in  the  various  regions  of  the 
United  States  shows  a high  degree  of  diversity.  In  one  region 
corn,  oats,  hay,  pasture,  dairy  cows  and  hogs,  constitute  the  prin- 
cipal enterprises  combined  upon  the  individual  farm.  In  an- 
other region  the  combination  is  the  same  with  the  exception  that 
more  emphasis  is  given  to  com  and  hogs,  and  beef  cattle  replace 
the  dairy  cows.  In  other  regions  the  live  stock  is  unimportant 
and  special  crops  as  wheat,  cotton,  or  cane,  stand  out  as  the 
dominant  enterprise,  while  in  still  other  regions,  the  grazing  c-f. 
cattle  or  sheep  becomes  the  principal  enterprise. 

These  variations  in  farm  organization  are  due  to  differences 
in  soil,  climate,  labor  supply,  market  relations,  etc.  The  explana- 
tion of  differences  in  types  of  farming  so  far  as  they  are  due  to 
differences  in  environment,  is  the  purpose  of  the  geographical 
method. 

The  geographical  method  may  be  illustrated  by  comparing 
maps  showing  the  distribution  of  spring  wheat  (Figure  10,) 
barley  (Figure  11)  and  oats  (Figure  12.)  These  crops  are 
usually  counted  competing  crops.  They  occupy  the  same  place  in 
the  system  of  crop  rotation  and  they  would  require, the  attention 
of  the  farmer  about  the  same  time  of  year  in  any  given  region. 
A study  of  the  maps  shows  a remarkably  distinct  division  of  ter- 
ritory between  these  crops.  In  Minnesota,  for  example,  wheat 
growing  extends  east  to  a line  drawn  north  and  south  through 
Northfield.  East  of  this  line  barley  is  the  dominant  spring  grain 
crop.  In  Wisconsin,  barley  production  is  concentrated  in  the 
east  central  counties  noted  for  their  high  grade  barley  for  brew- 
ing purposes. 

The  centers  where  oat  production  is  concentrated  are  found 
farther  south.  Central  Illinois  and  north-central  Iowa  stand  out 
as  regions  where  oat  production  holds  a highly  important  place 
r n the  farm. 

That  climate  is  one  factor  in  determining  this  division  of 
territory  is  obvious  from  the  nature  of  the  division.  These  crops 


114 


Wisconsin  Experiment  Station. 


Figure  10— Wheat.  One  dot  equals  50,000  Figure  11.— Barley.  One  dot  equals  100,000  Figure  12.— Oats.  One  dot  equals  100,000 

bushels.  bushels.  , bushels. 

"These  charts  show  the  geographical  division  of  territory  between  three  competing  crops  in  Iowa,  Minnesota,  Wisconsin,  and  parts  of  Illinois, 
Missouri,  North  Dakota,  and  South  Drkota  according  to  the  census  of  1900. 


Economics  in  Agricultural  Education  and  Research.  115 

difer  in  their  demands  for  moisture  as  weR  as  in  their  require- 
ments with  regard  to  temperature.  Soil  differences  are  said  to 
play  an  important  part  in  this  division  of  territory.  The  rela- 
tion of  the  barley  regions  to  barley  markets  is  in  itself  suggestive 
of  another  geographic  factor  which  should  be  considered. 

This  study  might  be  carried  further  by  the  use  of  charts  of 
the  various  other  enterprises  which  are  complementary  or  sup- 
plementary to  this  group  of  competing  crops.  Such  maps  would 
show  the  spring  wheat  region  to  be  a flax  region  and  the  oat 
regions  to  be  corn  regions,  etc.  This  method  followed  out  com- 
pletely with  maps  of  livestock,  as  well  as  crops,  would  show  the 
type  of  farming  in  any  agricultural  region.  (See  bulletins 
209  and  210  of  this  experiment  station  for  further  examples.) 

The  maps  showing  the  type  of  farming  should  be  accompanied 
with  maps  showing  the  topography,  the  soil,  the  length  of  the 
growing  season,,  the  temperature  during  the  growing  season,  the 
rainfall,  the  market,  the  agricultural  population,  the  manufac- 
turing population,  the  mining  population,  etc.,  all  of  which 
help  to  explain  the  types  of  farming  in  terms  of  geographical 
differences. 

The  Statistical  Method 

The  statistical  method  stands  for  quantitative  study.  Much  of 
our  knowledge  of  economic  forces  corresponds  to  qualitative 
analysis  in  chemistry.  The  force  is  noted  but  not  measured. 
The  statistical  and  accounting  methods  look  to  the  measurement 
of  forces,  thus  putting  the  work  on  a quantitative  basis.  In  the 
treatment  of  every  subject  and  in  the  use  of  all  other  methods, 
all  data  which  lend  themselves  to  counts  or  measurements  should 
be  treated  statistically. 

There  are  specialists  who  devote  themselves  to  the  collection 
and  the  tabulation  of  statistics.  Their  finished  product  is  raw 
material  for  the  agricultural  economist. 

Sources  of  Statistical  Data.  There  is  no  one  greater  source 
of  material  for  the  student  of  agricultural  economics  than  the 
reports  of  the  federal  census.  They  contain  the  only  comprehen- 
sive source  of  material  from  which  it  is  possible  to  ascertain 
the  type  of  farming  in  every  section  of  the  United  States.  This 
gives  the  basis  for  the  study  of  comparative  agriculture,  which  is 
one  of  the  best  methods  of  gaining  knowledge  of  the  economic 
forces  which  determine  the  actions  of  farmers. 


116 


Wisconsin  Experiment  Station. 


Not  only  the  type  of  farming,  but  also  the  economic  status  of 
the  farmer  is  shown  by  the  census  for  every  county  in  the  United 
States.  Both  the  white  and  the  colored  farmers  are  divided  into 
seven  classes,  based  upon  the  relation  they  sustain  to  the  land 
they  cultivate. 

The  census  reports  give  the  data  on  which  to  base  historical 
studies  of  the  changes  which  are  taking  place  in  the  type  of 
farming  and  in  the  status  of  the  farmer,  as  well  as  a basis  for 
comparative  study  at  a given  time. 

The  first  agricultural  census  was  taken  in  1840.  This  first 
census  of  agriculture  was  a crop  and  livestock  census.  In  1850, 
general  farm  data  were  added  and  other  improvements  have  been 
added  each  decade  since. 

In  1880,  tenure  statistics  were  added.  Thus  the  basis  for 
studying  changes  in  the  type  of  farming  extends  over  a period 
of  seventy  years  and  the  data  for  studying  changes  in  the  status 
of  the  farmer  are  available  for  thirty  years. 

Too  little  use  has  been  made  of  these  valuable  materials  in  the 
past.  Two  methods  which  the  writer  has  found  useful  in  the  uti- 
lization of  these  statistics  may  be  mentioned : 

The  system  of  mapping  already  illustrated  providing  a chart 
with  close  gradation  of  variation  in  density  for  each  fact  pres- 
ented in  the  census,  puts  the  materials  in  form  for  ready  com- 
parisons both  historical  and  geographical.  Not  only  does  the 
series  of  maps  show  the  changes  in  the  localization  of  each  line  of 
production  through  a series  of  years,  and  show  how  the  territory 
is  divided  between  the  various  lines  of  production  at  a given 
time,  but  indicates  also  the  way  in  which  the  different  lines  of 
production  are  combined  in  a given  district,  thus  showing  the 
type  of  farming  in  each  district. 

The  tabular  method  can  also  be  used  to  advantage  in  compar- 
ing types  of  farming.  A table  indicating  the  proportion  of  the 
improved  land  in  farms  devoted  to  each  crop  shows  the  utiliza- 
tion of  the  land.  The  character  of  the  crops,  and  the  relation 
between  the  numbers  of  livestock  kept  and  the  amount  of  feedable 
products  produced  gives  a clue  to  the  way  in  which  the  products 
are  utilized,  whether  sold  in  their  original  form  as  a product  of 
the  field,  or  used  as  a basis  of  the  livestock  industry.  While  a 
series  of  maps  based  upon  quantities  of  product  can  be  made  to 
cover  the  seventy  years,  the  statistics  of  crop  acreage,  which 
forms  a better  basis  for  showing  the  utilization  of  the  land,  were 
first  published  in  1880. 


Economics  in  Agricultural  Education  and  Research.  117 

In  making  use  of  the  census  materials,  the  original  statistics 
by  counties  are  of  first  importance.  The  descriptive  material 
l which  describes  the  quality  of  the  statistics  is  next  in  import- 
ance. The  text  which  is  intended  to  supplement  the  statistics, 
while  useful,  is  of  less  permanent  importance  than  the  detailed 
statistics. 

State  census  reports,  the  annual  reports,  and  more  recently, 
the  Year  Books  of  the  U.  S.  Department  of  Agriculture,  and  the 
state  agricultural  reports  Contain  valuable  statistics.  Market  re- 
ports in  trade  papers  and  in  reports  of  boards  of  trade,  etc.,  pro- 
vide statistics  of  use  in  the  study  of  prices.  While  these  various 
sources  are  not  all  that  may  be  desired,  the  student  will  find  that 
anything  he  can  do  for  himself  in  the  collection  and  tabulation 
of  statistics  will  be  but  a drop  in  the  bucket  in  comparison  with 
the  data  available  in  these  sources  which  have  been  prepared  by 
the  statistician.  Since  he  is  so  dependent  upon  these  sources  of 
material,  it  is  important  that  the  agricultural  economist  be  in 
I close  touch  witli  the  agricultural  statistician  who  is  preparing 
' these  source  books. 

The  use  of  market  statistics  may  be  illustrated  by  a study  of 
the  relation  of  the  price  of  corn  to  the  supply  and  price  of  hogs. 
The  causes  of  the  recent  high  prices  of  hogs  on  the  Chicago 
market  can  best  be  understood  by  studying  the  recent  history 
of  the  hog  and  corn  industries  as  shown  by  the  market  statistics. 
When  the  statistics  of  prices  are  studied  it  is  an  undeniable  fact 
that  hog  prices  were  higher  (Figure  13)  in  1910  than  at  any 
other  time  for  a long  series  of  years. 

The  general  theory  being  accepted  that  this  rise  in  price  must 
be  due  to  some  change  in  the  conditions  of  supply  or  the  condi- 
tions of  demand,  the  student  should  proceed  to  study  the  con- 
ditions of  supply  and  demand.  By  charting  the  supply  of  hogs 
on  the  Chicago  market  month  by  month,  the  fact  becomes  clear 
that  during  the  years  1909  and  1910  the  supply  of  hogs  was  un- 
usually short.  The  price  of  hogs  was  unusually  high  during 
the  same  period.  Little  time  is  required  to  make  the  inference. 
But  what  was  the  cause  of  the  shortage  of  hogs  ? One  may  pro- 
ceed to  formulate  hypotheses  which  may  be  put  into  the  form 
of  questions.  Has  some  pestilence  ravaged  the  hog  lots  of  the 
country?  Has  there  been  a failure  of  the  crops  which  are  used 
as  hog  feed?  Corn  being  the  principal  hog  feed,  we  may  ask, 
has  the  demand  for  corn  for  other  purposes  been  unusually  great? 


118 


Wisconsin  Experiment  Station. 


These  conjectures  as  to  the  cause  of  the  scarcity  of  hogs  leads 
to  the  charting  of  the  Chicago  price  of  corn. 

During  the  years  1906  and  1907  the  price  of  hogs  was  very 
high  in  proportion  to  the  price  of  corn.  Figure  13.  The  sup* 
ply  of  hogs  was  relatively  short  during  the  early  part  of  the 
period,  but  by  the  beginning  of  1908  the  supply  of  hogs  on  the 
Chicago  market  reached  an  unusually  high  mark.  During  the 
year  1908  the  corn  price  curve  held  steadily  and  appreciably 
above  the  hog  price  curve.  On  the  assumption  that  a bushel  of 
corn  is  required  to  produce  ten  pounds  of  pork,  this  meant  a 


Figure  13. — Relation  of  the  price  anti  supply  of  hogs  to  the  price  of  corn  at 

Chicago. 

The  price  curves  are  so  drawn  that  when  one  bushel  of  corn  produces  10  pounds  of 
pork,  the  amount  which  the  hog  price  curve  rises  above  the  corn  price  curve  repre- 
sents the  net  return  from  the  extra  labor  of  breeding  and  feeding.  In  1906  and 
1907  there  was  large  profit  in  feeding  corn  to  hogs,  while  in  1908  there  was  a loss. 
The  figures  on  the  margin  represent  the  monthly  high  price  of  corn  in  cents  per 
bushel;  the  monthly  high  price  of  hogs  in  tens  of  cents  per  pound  and  the  monthly 
receipts  of  hogs  in  tens  of  thousands. 

loss  on  the  corn  as  well  as  on  the  time  employed  in  feeding,  for 
ten  to  one  is  the  price  relation  in  the  chart.  The  distance  which 
the  curve  showing  hog  prices  rises  above  the  curve  showing  corn 
prices  shows  the  return  for  labor  in  feeding.  In  1908  this  was  a 
minus  quantity.  This  condition  had  not  existed  before  during 
the  past  fifteen  years.  Early  in  1909  the  curves  are  close  to- 
gether, but  the  corn  price  was  downward,  while  the  hog  price 


Economics  in  Agricultural  Education  and  Research.  119 


was  unusually  high.  Early  in  1910  hog  prices  started  down 
and  the  general  trend  has  been  downward  since  that  time. 

So  soon  as  the  situation  as  to  the  price  of  corn  and  hogs  and 
the  supply  of  hogs  since  1907  is  impressed  upon  the  mind,  it  is 
hard  to  keep  from  making  the  inference  that  because  hogs  were 
fed  at  a loss  in  1908  many  farmers  ceased  to  breed  hogs  in  the 
usual  numbers.  The  facts  indicate  that  whether  it  was  lack  of 
breeding  or  some  other  cause  the  supply  of  hogs  in  the  country 
was  short,  for  so  high  a price  for  so  many  months  would  cer- 
tainly stimulate  shipments  if  marketable  hogs  were  to  be  found. 

The  next  fact  requiring  explanation  is  the  high  price  of  corn 
in  1908.  A glance  at  the  curves  shows  that  during  the  greater 
part  of  the  year  the  hog  price  was  quite  normal,  but  the  corn 
price  was  abnormally  high. 

The  receipts  of  corn  on  the  Chicago  market  in  1908  were  34,- 
000,000  bushels  below  that  of  the  previous  year,  and  the  lowest 
they  had  been  since  1895,  with  the  exception  of  the  years  1901 
and  1902  when  the  receipts  were  low  and  the  price  high  owing 
to  the  short  corn  crop  of  1901.  But  why  this  shortage  in  re- 
ceipts of  corn  on  the  Chicago  market  in  1908? 

The  corn  crop  of  1907  was  less  than  it  had  been  for  the  two 
preceding  years,  but  with  the  exception  of  1905  and  1906  the 
crop  of  1907  was  the  largest  reported.  The  shortage  of  the  corn 
crop  of  1907  under  that  of  1906  was  only  11.5  per  cent,  but 
the  receipts  of  hogs  at  Chicago  during  the  period  when  the 
bulk  of  the  hogs  which  had  been  fed  1907  corn  were  marketed, 
indicate  that  out  of  this  relatively  small  corn  crop  of  1907  an 
unusually  large  number  of  hogs  were  fed  leaving  a relatively 
short  supply  for  the  market.  Before  drawing  conclusions,  how- 
ever, other  factors  need  be  carefully  investigated  to  make  sure 
that  other  important  forces  were  not  operating  simultaneously. 
These  facts  are  presented  in  this  form  in  order  to  illustrate  this 
well  known  method  of  putting  statistical  data  .in  such  form  that 
their  relations  are  easily  comprehended. 

The  Accounting  Method 

Cost  accounting  is  a method  of  ascertaining  such  facts  regard- 
ing the  operation  and  results  of  a business  as  will  enable  the 
operator  to  know  what  to  produce  and  how  to  produce  it  in 
order  to  secure  maximum  profits  from  the  business.  The  data 
secured  by  this  method  may  have  some  general  value,  but  its 


120 


Wisconsin  Experiment  Station. 


primary  purpose  is  to  give  a basis  for  more  intelligent  direction 
of  the  operations  of  the  factory  or  the  farm  for  which  the 
accounts  are  kept. 

Ihe  one  who  plans  the  records  and  their  tabulation  must  have 
a clear  vision  of  economic  forces  if  he  would  plan  a successful 
system  of  accounts,  for  economic  forces  determine  what  should 
be  done  on  the  farm.  The  system  of  accounts  must  show  quan- 
titatively the  workings  of  these  forces  at  a given  time  and  place. 

In  agricultural  accounting,  the  first  problem  is  to  contrive  a 
system  of  records  which  will  show  what  to  produce.  This  pro- 
blem is  more  complex  in  farming  than  in  almost  any 
other  business.  In  very  few  districts  does  the  farmer  devote 
himself  exclusively  to  one  enterprise.  This  is  not  due  to  any 
peculiar  characteristics  of  the  men  engaged  in  the  business,  but 
is  inherent  in  the  natural  conditions  under  which  crops  must  be 
grown.  There  are  more  or  less  definite  times  when  planting, 
harvesting,  etc.,  must  be  done,  and  it  is  rarely  found  that  one 
enterprise  such  as  wheat  growing  or  corn  growing  occupy  all 
the  farmer ’s  time. 

The  problem  of  first  importance  in  the  organization  of  a farm 
for  profits  is  that  of  correlating  a group  of  enterprises  upon  one 
farm  in  such  a manner  as  will  keep  the  labor  and  equipments 
employed  as  nearly  continuously  as  practical,  and  in  that  enter- 
prise which  will  yield  the  largest  returns  of  all  those  which  can 
be  carried  on  at  the  given  time  of  year. 

It  is  a matter  of  common  knowledge  that  the  production  of  a 
crop  of  corn  does  not  require  labor  continuously  throughout  the 
year.  The  same  is  true  of  oats  and  it  happens  that  the  nature 
of  these  plants  is  such  that  oats  can  and  must  be  sown  earlier 
than  corn,  and  that  oats  require  no  further  attention  until  after 
the  corn  has  been  planted  and  cultivated.  The  harvesting  and 
threshing  of  the  oats  are  or  can  be  over  before  the  corn  is  ready 
to  be  harvested.  These  crops  are,  for  these  reasons,  said  to  be 
complementary.  (Compare  Figures  15  and  18.) 

Corn  is  not  the  only  crop  which  requires  attention  at  tin' 
given  time  of  year.  In  some  regions,  tobacco  and  corn  are  both 
open  to  the  farmer’s  choice.  Some  places  the  sugar  beet  is  an 
alternative,  in  others,  cotton.  Likewise,  the  farmer  has  a choice 
of  several  crops  in  the  seasons  when  oats  are  sown  and  harvested. 
Barley  and  spring  wheat  will  suggest  themselves  as  alternatives 
to  oats  in  certain  regions.  The  crops  which  require,  attention  at 
the  same  time  of  year  are  said  to  be  competitive.  (Figures  18 
and  19.) 


Economics  in  Agricultural  Education  and  Research.  121 


It  is  obviously  to  the  farmer ’s  interest  that  he  select  from  each 
group  of  competing  crops  the  one  which  will  acid  most  to  his  net 
income.  It  is  equally  clear  that  he  will  desire  to  combine  as 
many  complementary  enterprises  as  will  add  to  the  profitableness 
of  the  business  as  a whole. 

The  problem  the  accountant  has  before  him  is  the  planning 
of  such  records  as  will  show  the  way  in  which  the  various  comple- 
mentary enterprises  fit  together  to  fill  out  the  year ’s  employment, 
and  such  records  as  will  enable  him  to  show  the  relative  profit- 
ableness of  each  of  the  competing  enterprises. 

To  secure  these  results  a labor  record  is  a necessity.  A labor 
record  showing  the  exact  distribution  of  all  man  and  horse  labor 
employed  each  day  in  the  year  gives  the  material  for  a chart 
which  will  show  the  time  employed  in  each  enterprise.  The 
charts  for  the  various  enterprises  on  one  farm  will  show  the 
complementary  character  of  certain  crops,  and  the  charts  for  a 
series  of  farms  in  the  same  locality  on  some  of  which  the  one,  on 
some  the  other,  of  a group  of  competing  crops  are  being  grown, 
the  competitive  character  of  certain  crops  cap  be  shown. 

It  is  necessary  to  know  what  was  done  and  what  might  have 
been  done  -in  order  to-judge  the  merits  of  the  management.  A 
labor  record  may  show  the  hired  man  was  cutting  wood  on  a 
given  forenoon  in  June.  If  supplementary  data  show  the  land 
was  too  wet  to  be  cultivated  and  that  the  clover  was  not  ready 
to  be  cut,  etc.,  it  may  prove  to  be  true  that  cutting  wood  was  the 
most  profitable  work  the  laborer  could  be  doing.  If,  however, 
in  July  the  records  show  a half  day  spent  in  repairing  a binder 
preliminary  to  commencing  grain  harvest,  which  time  might  have 
been  employed  in  cultivating  corn,  or  in  harvesting  hay  or  grain, 
the  student  of  the  problem  of  farm  management  will  have  a right 
to  question  the  wisdom  of  cutting  wood  that  day  in  June  which 
might  have  been  used  in  repairing  the  binder  and  thus  resulting 
m the  saving  of  valuable  time  in  July. 


The  problem  of  crop  selection  may  be  illustrated  and  further 
elaborated  by  the  study  of  Figures  14  to  19.  It  happens  that  the 
farmer  for  whom  these  records  were  tabulated,  produced  barley, 
oats,  spring  wheat,  hay,  corn,  and  tobacco.  When  the  field 
labor  on  this  farm  is  charted,  it  becomes  obvious  that  during 
May,  June  and  a part  of  July,  corn  and  tobacco  are  competitive. 
The  labor  is  some  days  on  the  one  crop,  some  days  on  the  other, 
and  knowing  the  character  of  the  crop,  it  is  fair  to  assume  that 


122 


Wisconsin  Experiment  Station. 


had  he  planted  more  corn  and  no  tobacco,  the  time  put  on  the 
tobacco  during  the  period  of  planting  and  cultivation  would 
have  been  put  on  the  corn.  (Figures  18  and  19.) 

When  the  three  small  grains  are  compared  with  corn  and  to- 
bacco, it  is  clear  that  they  are  sowed  before  the  time  for  planting 
com  or  tobacco.  Also  that  they  are  harvested  in  the  latter  part 
of  July,  apparently  after  the  tilled  crops  were  laid  by.  This  sug- 
gests that  these  small  grain  crops  are  complementary  to  the  til- 
led crops  from  the  standpoint  of  demand  for  man  and  horse 
labor.  It  is  known  also  that  these  groups  of  crops  are  comple- 
mentary from  the  standpoint  of  the  demands  of  a good  system 
of  crop  rotation.  The  small  grains  may  provide  nurse  crops  for 
grasses  and  legumes.  The  tilled  crops  clean  and  give  tilth  to  the 
land. 

In  studying  the  demands  of  the  various  crops  upon  the 
time  of  the  farmer,  operations  should  be  divided  into  two  classes : 
(1)  Those  which  must  be  done  within  very  narrow  limits  of  time, 
such  as  seeding  and  harvesting  of  small  grain,  the  planting  and 
cultivation  of  corn  or  tobacco,  and  (2)  those  which  can  be  done 
equally  well  at  any  time  through  a period  of  considerable  length, 
such  as  plowing  and  threshing.  Labor  of  the  former  class  should 
always  take  precedence  over  that  of  the  latter  class,  but  labor  of 
the  second  class  should  not  be  put  off  until  it  must  be  done  when 
labor  of  the  first  class  is  demanding  attention.  If  plowing  is 
left  too  long,  it  may  delay  the  planting;  if.  threshing  is  post- 
poned too  long,  it  may  conflict  with  tobacco  harvest  or  silage 
cutting. 

Passing  from  these  more  general  conditions  shown  by  the  chart 
to  the  further  details  of  each  of  these  groups,  it  is  interesting  to 
note  the  way  in  which  this  farmer  employed  his  labor  on  the 
three  spring-grain  crops.  The  wheat  and  the  oats  were  seeded 
and  harvested  so  nearly  at  the  same  time  that  they  appear  to  be 
strictly  competitive.  The  barley,  however,  is  sown  later  and 
harvested  earlier.  Barley  has  a shorter  season.  By  a proper 
combination  of  barley  with  the  other  spring  grains,  both  the  seed 
time  and  the  harvest  were  spread  over  a longer  period.  This 
might  have  been  accomplished  by  selecting  two  varieties  of  oats 
which  vary  in  the  length  of  their  period  of  growth. 

While  corn  and  tobacco  appear  to  be  competitive  during  the 
season  when  com  is  cultivated,  it  appears  that  they  are  comple- 
mentary in  their  demands  for  labor  in  the  later  operations.  The 


Economics  in  Agricultural  Education  and  Research.  123 


cutting  of  the  tobacco  preceded  the  cutting  of  the  corn  and  the 
husking  or  shredding  of  the  corn  preceded  the  stripping  of  the 
tobacco.  Had  the  corn  been  cut  for  silage,  the  harvesting  of 
the  corn  and  the  tobacco  might  have  demanded  labor  at  the 
same  time. 

Hay  harvest  took  the  labor  of  the  farm  from  the  corn  and  to- 
bacco fields  for  about  a week  at  the  end  of  June.  Hay  crops 
differ  with  respect  to  the  time  they  must  be  harvested.  The 
later  in  the  season  of  corn  cultivation  the  hay  harvest  comes,  the 
less  serious  is  the  conflict  with  the  corn  crop.  The  cultivation 
of  corn  when  it  is  small  is  a slower  process  than  when  it  has  at- 
tained a height  of  a foot  or  more.  The  amount  of  corn  one  can 
cultivate  when  it  is  small,  sets  a limit  to  the  size  of  the  corn  crop. 
One  can  cultivate  this  amount  at  the  later  stages  and  have  time 
left  for  making  hay.  Hence  the  hay  harvest  may  come  at  a time 
when  it  can  be  counted  complementary  to  corn.  A study  should 
be  made  of  alfalfa,  clover,  and  other  hay  crops  with  respect  to 
the  way  in  which  they  can  be  fitted  into  a system  of  complemen- 
tary enterprises.  If  for  example  it  should  be  shown  that  the 
first  cutting  of  alfalfa  must  be  harvested  just  at  the  time  when 
corn  demands  its  first  cultivation,  it  would  become  obvious  that 
to  increase  the  alfalfa  crop  will  necessitate  a reduction  of  corn. 
The  question  then  would  be,  “ Which  of  these  crops  adds  most 
to  the  profits  of  the  farm?” 

The  next  step  in  the  study,  is  to  compare  the  relative  profit- 
ableness of  the  competing  crops.  The  accompanying  table  illus- 
trates methods  of  comparing  relative  profitableness. 


Methods  of  Comparing  Profits* 


Profit  Profit  per 

Crop.  per  acre  hour  of  labor 

Barley  $12.75  $ .734 

Oats  13.97  .785 

Wheat  19.72  .969 

Hay  14.16  1.868 

Corn  ; 6.82  .371 

Tobacco  ’ 18.59  .093 


* Based  upon  records  secured  in  co-operation  with  United  States 
Department  of  Agriculture. 


124 


Wisconsin  Experiment  Station. 


These  figures  are  for  the  same  farm  for  which  the  labor  dis- 
tribution is  shown  in  Figures  14  to  19.  In  calculating  profits 
on  the  specific  crops  the  general  farm  expenses  were  not  con- 
sidered. It  is  relative  profitableness,  not  the  absolute  net  profit 
which  is  compared. 

No  generalization  can  be  drawn  from  this  table  as  to  which 
crops  will,  as  a rule,  be  most  profitable  to  the  farmer.  It  hap- 
pened that  the  small  grain  crops  were  good  and  the  corn  crop 
generally  poor  the  year  these  records  were  kept.  It  happened 
also  that  half  the  corn  and  all  the  tobacco  had  to  be  planted  a sec- 
ond time  which  increased  the  labor  cost  of  these  crops.  But  this 
table  is  of  significance  in  that  it  contains  evidence  that  the  farm- 
er or  the  experiment  station  worker  is  in  danger  of  going  wrong 
if  he  applies  generally  the  common  method  of  comparing  profits 
per  acre.  On  the  basis  of  profit  per  acre,  the  tobacco  was  al- 
most three  times  as  profitable  as  the  corn,  but  on  the  basis  of 
profit  per  hour,  the  corn  was  about  four  times  as  profitable  as  the 
tobacco.  Where  approximately  the  same  labor  is  expended  per 
acre  the  same  result  is  reached  whether  one  uses  the  acre  or  the 
labor  basis.  As  has  been  suggested  on  page  95,  profit  per  acre 
multiplied  by  the  number  of  acres  the  farmer  can  handle  of  the 
two  crops  may  be  a means  of  combining  the  acre  and  the  labor 
basis  of  calculating  relative  profits. 

The  relative  merits  of  these  methods  will  not  bo  discussed  here. 
All  methods  should  be  tested  and  a search  made  for  the  bed 
possible  plan  for  comparing  the  profitableness  of  crops, 

Taking  profits  per  unit  of  labor  as  a starting  point  for  further 
consideration,  note  some  of  the  limitations  and  complexities  in- 
volved in  its  use.  Where  two  crops  can  be  found  which  require 
the  attention  of  the  farmer  at  exactly  the  same  time  throughout 
all  their  operations  and  in  forms  of  labor  which  require  the  same 
amount  of  managerial  activity  per  unit  of  labor  the  question  of 
relative  profitableness  is  easily  worked  out  on  the  basis  of  profit 
per  unit  of  labor,  but  where  crops  are  competitive  for  a portion 
of  the  year  and  complementary  for  the  remainder  of  the  year, 
the  solution  of  the  problem  of  relative  profitableness  is  not  so 
simple. 

Corn  and  tobacco  give  a good  example.  These  crops  are  com- 
petitive at  the  stages  when  it  is  vital  that  the  work  be  done  with- 
out delay.  This  means  that  the  one  crop  cannot  be  increased 
without  decreasing  the  other.  Yet  a very  large  proportion  of  the 


Figure  14. — Distribution  of  man  labor  on  14.8  acres  of  barley  In  1910  showing  the  time  of  year  when  barley  demanded  the  time  of  the  farmer  who  de- 
voted 17.4  hours  of  man  labor  per  acre  to  its  production.  The  profit  per  acre  was  $13.05  and  the  profit  per  hour  of  man  labor  75  cents. 


Economics  in  Agricultural  Education  and  Research.  125 


labor  on  these  crops  can  be  performed,  as  shown  in  Figures  18 
and  19  without  any  conflict  of  one  with  the  other.  Before  any 
conclusion  can  be  drawn  as  to  which  of  these  crops  yields  the- 
larger  return  per  unit  of  labor,  it  is  necessary  to  ascertain  to  what, 
alternative  use  the  labor  could  have  been  put  and  the  rate  of  re- 
turn the  labor  would  have  yielded  during  the  time  devoted  to 
harvesting  and  stripping  the  tobacco  and  the  time  of  harvesting 
and  shredding  the  corn.  Using  this  rate  as  the  “opportunity 
cost”  of  the  labor  at  these  periods  and  charging  it  against  the 
crop  using  the  labor  at  these  times  of  non-competitive  use,  the 
remainder  of  the  net  return  to  labor  can  all  be  accredited  to  the 
labor  during  the  period  of  competitive  use.  The  crop  which, 
by  this  method,  shows  in  the  long  run  the  highest  return  to  labor 
during  the  competitive  period  will  prove  the  more  profitable. 

While  the  above  illustrations  relating  to  what  to  produce  deal 
with  crop  selection,  the  problem  of  whether  to  sell  or  feed  the 
feedable  products  of  the  farm,  and  the  problem  of  the  kind  of 
livestock  to  keep  are  equally  important.  The  attention  of  a 
skillful  accountant  is  required  to  plan  a system  of  records  which 
will  give  basis  for  passing  judgment  on  these  questions. 

Experiments 

Another  set  of  problems  which  the  agricultural  economist 
may  well  consider,  relates  to  the  proportions  in  which  the  factors 
of  production  should  be  combined.  The  most  familiar  phase  of 
this  question  is  commonly  spoken  of  as  '/intensity  of  culture.” 
Intensity  of  culture  relates  to  the  proportions  in  which  land  and 
the  other  factors  of  production,  labor  and  capital,  are  combined. 
Other  questions  of  proportions,  as  the  relation  of  man  to  horse 
labor,  the  relation  of  investment  in  food  and  in  shelter  for  live- 
stock, etc.,  are  equally  important,  but  for  purposes  of  illustration, 
the  problem  of  intensity  of  culture  may  best  serve  the  purpose. 
The  proper  degree  of  intensity  of  culture  is  becoming  of  great 
importance  for  the  simple  reason  that  increasing  land  values  de- 
mand a higher  degree  of  intensity  of  culture  than  was  justifiable 
formerly  when  land  values  were  lower.  Unfortunately  the  de- 
gree of  intensity  which  was  formerly  justifiable  has  become  more 
or  less  definitely  established  by  custom  and  considerable  effort 
will  be  required  to  induce  farmers  to  leave  the  customary  mode  o f 
farming  for  a more  intensive  culture. 


126 


Wisconsin  Experiment  Station. 


In  order  to  carry  on  investigations  in  intensity  of  culture,  the 
experimental  method  must  be  made  use  of.  Some  of  the  data 
essential  to  the  analysis  of  the  economic  problems  of  the  farm  can 
be  secured  by  keeping  records  upon  farms  under  the  manage- 
ment of  intelligent  farmers  with  whom  it  is  possible  to  cooperate. 
By  this  method,  the  problem  of  combining  complementary  enter- 
prises in  such  a manner  as  will  keep  the  labor  and  equipment 
employed  as  nearly  continuously  as  possible  and  in  the  lines  cf 
production  which  will  prove  most  profitable,  can  be  worked  out 
in  a manner  fairly  satisfactory.  Furthermore,  the  contact  with 
the  farm  under  conditions  of  normal  commercial  agriculture 
gives  validity  to  the  results  secured  and  gives  the  opportunity 
for  developing  a system  of  records  which  may  ultimately  be  used 
by  any  intelligent  farmer  in  determining  what  to  produce. 

When  one  turns  from  the  question  of  what  to  produce  to  the 
question  of  how  it  should  be  produced,  the  problem  becomes  one 
which  requires  controlled  experiments.  The  major  economic 
problem  relating  to  the  question  of  how  to  produce  the  articles 
decided  upon,  centers  in  the  question  of  the  proportions  in  which 
the  factors  of  production  shall  be  utilized,  the  best  known  phase 
of  which  is  the  problem  of  intensity  of  culture.  This  is  a ques- 
tion regarding  which  agriculturists,  economists  and  farmers  have 
theorized  for  centuries,  but  regarding  which  no  adequate  ex- 
periments have  been  carried  out.  In  the  theoretical  analysis  of 
this  problem,  the  point  has  been  reached  where  experiments  are 
essential  to  further  progress. 

The  proper  degree  of  intensity  of  culture  must  be  determined 
for  each  farm,  and  the  result  will  change  with  variation  in  the 
wages  of  labor,  the  cost  of  equipment,  and  the  price  of  land. 
The  first  step  toward  progress  in  this  line  is  the  discovery  of  a 
method  of  experimentation  which  can  be  applied  upon  any  farm 
without  state  aid  and  without  endangering  the  profits  of  the 
farmer. 

Experiments  with  a series  of  plots  with  varying  treatment  are 
valuable  for  ascertaining  physical  and  biological  truths,  but  it 
is  doubtful  if  they  are  of  use  in  the  field  of  economics  for  the 
simple  reason  that  while  the  laws  of  economics  which  determine 
the  proper  degree  of  intensity  of  culture  are  of  general  applica- 
tion, the  conditions  are  so  variable  that  the  proper  degree  of  in- 
tensity on  one  farm  is  not  necessarily  the  proper  degree  on 
another.  Plot  experiments  on  the  intensity  of  culture  would 


Economics  in  Agricultural  Education  and  Research.  127 


have  no  practical  value  therefore  except  for  the  farm  on  which 
they  were  made.  Plot  experiments  are  too  expensive  for  the 
practical  farmer,  hence  some  other  method  must  be  contrived. 
It  is  highly  desirable  that  a method  of  ascertaining  the  proper 
degree  of  intensity  of  culture  be  discovered  and  taught.  Any 
attempt  at  teaching  more  than  the  principles  involved  and  the 
methods  of  their  application  is  folly,  for  what  is  right  for  one 
member  of  a class  of  one  hundred  students,  or  an  audience  of 
farmers,  is  not  likely  tp  be  correct  practice  for  many  of  the 
others. 

Differences  in  the  soil,  in  the  value  of  the  land,  in  the  effi- 
ciency of  the  farmers,  and  in  facilities  for  marketing  make  dif- 
fering degrees  of  intensity  of  culture  necessary.  A method  of 
ascertaining  the  most  profitable  degree  of  intensity  on  any  farm 
is  much  needed.  To  be  of  general  use  it  must  be  so  planned 
that  its  application  will  not  endanger  the  profits  of  the  farm. 
Some  process  of  gradual  adjustment  suggests  itself  as  most  like- 
ly to  succeed. 

The  foregoing  discussion  is  not  intended  as  a complete  survey 
of  methods  applicable  to  the  study  of  economic  problems  in 
agriculture.  The  aim  has  been  simply  to  describe  the  methods 
in  use  at  this  station  at  the  present  time.  No  one  method  is 
favored  above  another.  All  are  needed  in  securing  an  intensive 
and  a comprehensive  view  of  the  economic  forces  which  affect 
the  farmer. 


128 


Wisconsin  Experiment  Station. 


APPENDIX 

CHAPTER  HEADINGS  OF  A COURSE  IN  AGRICULTURAL 
ECONOMICS. 

Chapter  I.  Agricultural  economics  defined  and  described. 

Chapter  II.  The  economic  characteristics  of  the'  land  basis  of 
agriculture. 

Chapter  III.  The  economic  characteristics  of  the  human  basis 
of  agriculture. 

Chapter  IY.  Farm  equipment. 

Chapter  Y.  The  grades  of  the  factors  of  production,  and  the 
combination  of  these  grades. 

Chapter  YI.  The  divisions  of  occupations,  the  division  of  labor, 
and  the  combination  of  enterprises. 

Chapter  YII.  The  choice  of  crops,  the  planning  of  the  rotation. 

Chapter  VIII.  The  place  of  animal  husbandry  in  the  economy 
of  the  farm. 

Chapter  IX.  Manufactures  on  the  farm. 

Chapter  X.  The  proportion  in  which  the  factors  of  agricultural 
production  should  be  combined. 

Chapter  XI.  The  size  of  farms. 

Chapter  XII.  Methods  of  ascertaining  the  relative  profitable- 
ness of  the  different  forms  of  farm  organization. 

Chapter  XIII.  Systems  of  land  tenure  viewed  from  the  stand- 
point of  production. 

Chapter  XIY.  Labor  systems  viewed  from  the  standpoint  of 
production. 

Chapter  XY.  The  influence  of  credit  systems  upon  the  organ- 
ization of  agricultural  production. 

Chapter  XYI.  Agricultural  production  from  the  national  view- 
point. 

Chapter  XVII.  The  laws  of  demand. 

Chapter  XVIII.  The  theory  of  value. 

Chapter  XIX.  Monopolies  and  monopoly  prices. 

Chapter  XX.  Money. 

Chapter  XXI.  Credit  and  banking. 

Chapter  XXII.  The  economy  of  good  roads. 

Chapter  XXIII.  Railway  transportation. 


Economics  in  Agricultural  Education  and  Research.  129 


■Chapter  XXIY.  Opportunities  for  direct  sale  of  farm  products. 

Chapter  XXY.  Sales  to  or  through  middlemen. 

Chapter  XXYI.  Cooperation  and  concerted  action  on  the  part 
of  farmers  in  marketing  their  products. 

Chapter  XX YII.  Prices  and  the  marketing  of  cereals. 

Chapter  XXYIII.  Prices  and  the  marketing  of  cotton. 

Chapter  XXIX.  Prices  and  the  marketing  of  tobacco. 

Chapter  XXX.  The  sugar  supply  and  the  price  of  sugar. 

Chapter  XXXI.  Prices  and  the  marketing  of  live  stock. 

Chapter  XXXII.  Prices  and  the  marketing  of  dairy  and  poul- 
try products. 

Chapter  XXXIII.  Prices  and  the  marketing  of  wool. 

Chapter  XXXIY.  Prices  and  the  marketing  of  fruits  and 
vegetables. 

Chapter  XXXY.  A comparative  study  of  the  prices  of  farm 
products. 

Chapter  XXXYI.  Government  in  its  relation  to  prices  and  the 
marketing  of  farm  products. 

Chapter  XXXYII.  The  prices  of  and  the  methods  oLlxbtaining ' 
goods  to  be  used  or  consumed  on  the  farm. 

Chapter  XXXYIII.  Monopolies  and  their  influence  upon  the 
prices  of  goods  which  farmers  buy. 

Chapter  XXXIX.  The  Government  in  its  relation  to  commerce 
in  household  supplies  and  farm  equipments. 

Chapter  XL.  The  general  law  of  wages. 

Chapter  XLI. . The  farm  labor  supply  and  wages  from  the 
farmer’s  viewpoint. 

Chapter  XLII.  Employment  and  wages  from  the  laborer’s 
viewpoint. 

Chapter  XLIII.  Rent. 

Chapter  XLIY.  The  farmer’s  profits. 

Chapter  XLY.  The  rate  of  interest. 

Chapter  XLYI.  The  price  of  land. 

Chapter  XLYII.  The  means  of  acquiring  knd. 

Chapter  XLYIII.  The  economic  status  of  the  agricultural 
classes. 

Chapter  XLIX.  Opportunities  in  agriculture  compared  with 
those  in  other  occupations. 

Chapter  L.  The  Government  in  its  relation  to  property  rights, 
contracts,  etc. 

Chapter  LI.  The  farmer’s  interest  in  public  expenditures. 


130 


Wisconsin  Experiment  Station. 


Chapter  LII.  The  farmer’s  interest  in  the  various  forms  of 
taxation. 

Chapter  LIII.  Who  pays  the  tax — the  “incidence”  and  the 
influence  of  taxation. 

Chapter  LIV.  Revenues  from  productive  property  owned  or 
operated  by  the  Government. 


'!1 

Physiological  Effect  on  Growth  and  Repro- 
duction of  Rations  Balanced  from 
Restricted  Sources 


E.  li.  HART,  E.  V.  McCOLLUM,  H.  STEENBOCK,  AND 
G.  C.  HUMPHREY.* 

Scientific  studies  of  the  nutrition  of  farm  animals,  up  to 
the  present  time,  have  been  directed  almost  wholly  toward  the 
problem  of  income  or  outgo  of  matter  or  energy,  or  both. 
The  early  development  of  the  science  of  animal  nutrition 
largely  took  the  form  of  establishing  in  a practical  way  what 
should  be  the  necessary  supply  of  digestible  nutrients,  includ- 
ing proteins,  fats  and  carbohydrates  for  the  different  purposes 
of  performance  to  which  the  domestic  animal  was  subjected. 
This  method  was  introduced  by  Htenneberg  about  3860  and 
led  to  the  development  of  standards,  notably  those  of  Wolff, 
which  liave  seen  large  use.  These  standards,  have  been  subject 
to  some  modification  from  time  to  time  as  more  practical  ex- 
perience and  more  definite  chemical  data  on  the  composition 
of  feeds  and  their  co-efficients  of  digestibility  accumulated. 
However,  no  important  modification  in  the  methods  of  investi- 
gation upon  which  these  standards  are  founded,  was  made 
.until  the  introduction  of  the  method  for  measuring  the  energy 
of  feeding  materials  and  the  gaseous  exchange  of  the  animal, 
as  well  as  the  income  and  outgo  of  energy  from  the  body  by 
means  of  the  respiration  calorimeter.  This  latter  method  of 
investigation  made  possible  the  measurements  of  income  and 
outgo  of  both  matter  and  energy.  By  it,  the  production  value 


* This  investigation  has  been  cooperative  between  the  departments 
of  Agricultural  Chemistry  and  Animal  Husbandry.  Professors  Hart, 
McCollum  and  Mr.  Steenbock  were  in  charge  of  the  plan  and  chemical 
side  of  the  work.  Prof.  Humphrey  has  been  responsible  for  the  feed* 
ing  and  care  of  the  animals. 


132  Wisconsin  Experiment  Station. 

or  relative  capacity;  of  feeds  to  produce  increase  of  animal 
product  is  measured.  The  development  of  this  method  of 
work,  so  far  as  the  domestic  animal  is  concerned,  has  been 
made  principally  by  Rubner,  Zuntz,  Kellner,  Hagemann  and 
Armsby.  The  outgrowth  of  this  type  of  work  has  resulted  in 
the  construction  .of  modified  standards,  particularly  those  of 
Kellner  and  Armsby,  for  feeding  domestic  animals.  While  at 
present  incomplete  it  is  expected  that  with  the  accumulation 
of  more  data  their  formulation  for  all  classes  of  farm  animals 
will  be  extended  and  placed  upon  an  experimental  basis. 

All  the  above  methods  of  investigation  have  led  to  the  es- 
tablishment of  formulae  of  mathematical  expression.  It  has 
been  assumed  that  a proper  supply  of  nutrients  (carbohydrates, 
fats  and  proteins),  with  a proper  available  energy  intake  will 
subserve  all  the  animal’s  requirements  for  growth  and  repro- 
duction. Such  standards  expressed  mathematically  will  always 
be  of  value  because  of  their  definite  form,  and  it  is  just  such 
definite  information  that  the  practical  feeder  wants.  Probably 
none  have  felt  the  limitation  of  mathematically  constructed 
feeding  standards  more  than  those  who  have  taken  a promi- 
nent part  in  their  development,  and  even  the  practical  and 
successful  feeder  uses  these  standards  only  as  a help,  varying 
the  kind,  as  well  as  the  proportion,  of  total  nutrients  in  the 
ration  to  mefet  the  requirements  of  the  individual.  The  kind  of 
nutrients,  however,  receives  his  attention  only  when  their  ef- 
fects are  extremely  pronounced  and  immediately  apparent. 

But  in  addition  to  the  limitation  of  mathematical  stand- 
ards, which  consider  only  the  total  digestible  nutrients,  or  the 
total  net  available  energy  of  a ration,  there  are  still  other  im- 
portant factors  that  must  be  considered.  We  refer  to  what 
may  be  called  the  physiological  value  of  the  ration.  • 

The  rations  ordinarily  fed  our  farm  animals  are  exceed- 
ingly complex  in  chemical  composition.  There  are  many  dif- 
ferent proteins,  in  addition  to  nitrogen-bearing  bodies  of  non- 
protein character;  fats  of  different  composition  and  degree  of 
saturation;  carbohydrates  of  many  types;  and  almost  a host 
of  undetermined  and  undefined  bodies  in  the  daily  ration  of 
a domestic  animal.  Whether  this  complex  organic  ensemble 
of  the  farm  ration  is  always  conducive  to  vigorous  growth 
and  sustained  vitality,  or  whether,  dependent  upon  its  source, 
it  may  contain  either  nutrients  of  inadequate  chemical  consti- 


Effect  of  Rations  Balanced  from  Restricted  Sources.  133 


tution  or  depressants,  which  counteract  the  favorable  physi- 
ological effect  of  a part  of  the  ration,  is  an  unsolved  problem. 
It  is  true  that  practical  experience  soon  accepts  or  rejects  a 
* plant  material  proposed  for  animal  nutrition  on  the  basis  of  its 
“ agreeing' ’ or  “disagreeing”  with  the  animal.  But  unless 
the  effects  are  exaggerated  and  decisive,  it  may  either  be  im- 
mediately discarded  or  pass  into  use  and  become  a part  of  the 
category  of  utilizable  feeds.  Cottonseed  meal  is  an  example 
in  point  and  is  a material  that  must  be  used  with  caution, 
especially  in  feeding  young  animals. 

In  addition  to  the  direct  relation  of  the  organic  portion  of 
the  ‘ration  to  physiological  vigor,  attention  must  also  be  directed 
to  another  phase  of  nutrition  problems  to  which  little  attention 
has  been  given.  We  refer  to  the  proportion  of  acid  radicals 
to  bases  in  the  feed  as  affecting  the  reaction  in  the  body  and 
indirectly  influencing  the  normal  physiological  processes.  Un- 
questionably the  physiological  value  of  a ration  is  largely  de- 
pendent'upon  its  chemical  constituents,  but  the  usual  determin- 
ations made  on  feeding  materials  do  not  reveal  the  character  or 
manner  of  combination  of  many  of  the  constituents.  Conse- 
quently the  physiological  value  can  be  determined  in  the  pres- 
ent state  of  our  knowledge,  only  by  long  continued  observations 
of  the  reaction  of  the  feed  on  the  animal. 

With  the  recognition  of  the  possibility  of  there  being  a 
physiological  value  to  the  ration  not  determinable  by  total 
intake  of  digestible  nutrients,  nor  by  a measurement  of  its 
production  energy  value,  a plan  of  investigation  designed  to 
throw  some  light  on  this  problem  was  inaugurated. 

Certain  phases  of  this  subject  of  the  relation  of  food  to  metabol- 
ism and  resistance  to  poisons  have  already  received  some  at- 
tention. The  work  of  Hunt1  on  the  resistance  of  mice,  fed 
different  diets  to  acetonitrile  is  of  especial  interest  in  this  con- 
nection. Poster 2 has  also  studied  the  effect  of  diet  on  the 
resistance  of  dogs  to  ricin ; the  influence  of  diet  on  the  develop- 
ment of  certain  glands  has  been  studied  by  Watson  3,  who  re- 
ports that  the  thyroid  of  rats  is  enlarged  by  an  excessive  oatmeal 
diet.  The  work  of  Pugliese  and  Brighenti  4 revives  again  the 

1 Bui.  69,  Hyg.  Lab.,  U.  S.  Treasury  Dept. 

2 Proc.  Soc.  Exp.  Biol.  & Med.,  1909,  6,  p.  61. 

3 Lancet,  1907,  1,  p.  985. 

4 Jour,  de  Physiol,  et  de  la  Pathol,  gen,  1909,  11,  p.  1047. 


134 


Wisconsin  Experiment  Station. 


idea  that  oats  have  a “stimulating”  action  upon  animals.  They 
found  that  the  products  of  autolysis  of  oats  and  of  their  peptic 
digestion  increase  the  strength  of  the  contraction  of  the  heart 
and  of  striated  muscle  and  delay  the  fatigue  of  the  latter. 

To  specific  constituents  of  foods  have  been  attributed  func- 
tions of  a positive  character.  The  relation  of  lecithin  to  growth 
has  received  much  attention  and  is  believed  by  some  to  have 
specific  effects.5 

The  statement  that  the  proteins  of  a ration  have  specific 
dynamic  functions  apart  from  all  calorific  and  constructional 
value,  is  also  believed  by  students  of  nutrition. G 

In  the  progress  of  our  own  work,  which  up  to  the  present  time 
has  been  conducted  entirely  upon  young  heifers  and  milch 
cows,  it  became  apparent  that  normal  rations  compounded 
from  different  sources  wTere  not  of  the  same  physiological  value. 
The  details  of  these  effects  will  be  fully  discussed  in  the  ex- 
perimental part  of  this  bulletin,  but  the  results  are  so  decisive 
that  their  bearing  on  the  whole  subject  of  both  human  and 
animal  nutrition  cannot  he  ignored.  Unfortunately  it  is  im- 
possible to  conduct  such  experiments  as  here  detailed  on  the 
human  family  involving  as  it  does  the  use  of  a special  ration 
for  a long  time  and  the  influence  of  such  a ration  on  the  milk 
producing  capacity  of  the  mother  and  the  size  and  vigor  of 
the  young.  But  such  specific  and  experimentally  established 
information  is  to-day  lacking  in  both  fields  of  human  and  ani- 
mal nutrition.  The  more  limited  effect  of  a ration  on  the  rate 
of  growth  as  compared  with  its  possible  effect  on  the  offspring 
emphasizes  the  absolute  necessity  of  continuing  through  the 
growing  period  of  the  animal  and  at  least  through  one  gesta- 
tion period  any  nutrition  experiment  designed  to  test  the  spe- 
cific physiological  value  of  a food  material. 

While  the  use  of  dietetic  treatments  in  certain  human  diseases 
of  the  alimentary  canal  has  given  satisfactory  results,  never- 
theless it  cannot  be  claimed  that  the  choice  of  various  foods 
in  health  and  disease  rests  upon  even  a theoretical  basis,  not  to 
say  an  experimental  basis. 


r>  Hatai,  Amer.  Jour,  of  pliysiol.,  1903,  10,  p.  57. 
6 Lusk,  Science  of  Nutrition,  p.  126. 


Effect  of  Rations  Balanced  from  Restricted  Sources.  135 


Plan  of  Experiment 

Experiments  of  a type  designed  to  answer  these  physiological 
questions  must  be  conducted  with  a considerable  number  of 
animals  and  over  long  periods  of  time.  Short  periods  of  ex- 
perimentation are  absolutely  useless  and  can  give  no  definite 
and  conclusive  answer  to  the  questions  involved.  By  long  periods 
is  meant  such  periods  of  time  as  will  cover  the  growth  of  the 
animal  to  maturity  and  reproduction.  Not  only  should  the  time 
of  feeding  specific  rations  be  of  long  duration,  but  the  animal 
should  be  subjected  to  all  the  strains  normally  placed  upon  the 
female.  The  strains  of  reproduction  and  milk  secretion  should 
always  be  included  in  the  experimental  regime. 

Animals  For  our  experiment,  sixteen  grade  Shorthorn  heifer 
calves  were  purchased  on  the  market.  One  of  the  animals  had 
an  admixture  of  Jersey  blood,  while  another  was  partly  Guern- 
sey. Heifer  calves  were  chosen  because  of  their  relation  to 
the  dairy  industry.  Parenthetically  it  may  be  stated  that 
results  secured  on  the  effect  of  rations  on  physiological  vigor 
with  one  species  may  not  with  safety  be  translated  in  the 
same  terms  for  another  species.  The  results  secured  here  are 
probably  only  applicable  to  this  particular  species  of  farm 
animals. 

The  heifers  were  divided  into  four  groups  of  as  nearly 
equal  total  weight  as  possible.  They  weighed  from  310  to  413 
pounds  and  were  approximately  five  to  six  moths  old  So  far  as 
judgment  could  be  exercised,  the  lots  were  as  nearly  alike  in  all 
respects ' as  to  health,  vigor  and  wTeight,  as  it  was  possible  to 
make  them. 

Feeds  The  source  of  nutrients  for  each  group  was  to  be 
limited  to  a single  plant.  It  is  now  definitely  known  that 
there  are  marked  variations  in  the  chemical  construction  of 
individual  proteins  of  the  cereal  grains.  The  difference  be- 
tween the  zein  of  the  corn  kernel  and  the  glutenin  of  the  wheat 
kernel  is  too  well  known  to  need  discussion.  Further,  from  un- 
published data  there  is  reason  to  believe  that  the  hydrolysis  of 
the  total  assembled  proteins  of  the  cereal  grains  involved  in 
this  research  will  yield  different  proportions  of  amino  acids. 
But  little  is  absolutely  known  concerning  the  nature  of  the 
nitrogenous  portion  of  the  coarse  feeds  used. 


336 


Wisconsin  Experiment  Station. 


Great  stress  in  modern  theories  of  nutrition,  has  been  laid 
upon  the  chemical  differences  in  the  proteins  common  to  our 
feeds,  with  their  possible  relation  to  problems  of  proper  nu- 
trition. No  such  consideration  has  been  given  to  the  possible 
differences  in  the  carbohydrates  and  fats  of  our  common  feeds. 
While  it  is  true  that  the  fats  of  cereal  grains  have  been  in- 
vestigated by  Stellwag  and  others,  and  the  carbohydrates  very 
extensively  by  Schulze  and  his  pupils,  so  far  as  we  are  aware 
no  importance  has  been  attached  to  these  differences  in  re- 
lation to  the  nutrition  problems  of  higher  animals.  It  may 
be  said,  however,  that  such  constitutional  differences  as  do  exist 
between  the  latter  two  classes  of  nutrients  from  different  sources 
are  probably  of  less  importance  than  in  the  case  of  the  pro- 
teins. 

To  the  ash  side  of  rations  some  attention  has  been  given, 
but  this  important  field  needs  closer  investigation  by  students 
of  animal  nutrition. 

With  the  possible  physiological  influence  of  nutrients,  dif- 
fering in  structure  and  forms  of  combination,  when  fed  over 
very  long  periods  of  time,  in  mind,  it  was  planned  in  initiating 
our  experiment  to  first  satisfy  the  requirements  of  the  Wolff 
standard.  This  requires  a supply  of  equal  quantities  of  digest- 
ible nutrients.  It  implies  a chemically  balanced  ration,  one  in 
which  the  gross  quantity  of  nutrients  is  alike,  but  not  necessar- 
ily of  the  same  chemical  character,  when  the  rations  are  de- 
rived from  various  plant  sources. 

Lot  I For  our  first  experiment  this  ration  was  made,  up  en- 
tirely from  the  nutrients  of  the  corn  plant.  To  secure  a fairly 
efficient  protein  intake,  the  ration  was  made  from  corn  stover, 
com  meal,  and  gluten  feed  (a  corn  by-product  of  high  protein 
content). 

Lot  II  was  to  receive  all  of  its  nutrients  from  thie  wheat 
plant.  This  ration  consisted  of  wheat  straw,  wheat  meal,  and 
wheat  gluten  (a  high  protein  wheat  by-product  procured  from 
the  Pure  Gluten  Feed  Company). 

Lot  III  was  to  receive  its  nutrients  from  the  oat  plant.  This 
ration  consisted  of  oat  straw  and  rolled  oats.  It  was  necessary 
to  make  the  protein  content  of  this  ration  the  standard  for  all 
other  lots,  since  no  oat  by-product  of  higher  protein  content 
than,  that  of  rolled  oats  can  be  purchased  on  the  market.  This 
ration  really  fixed  the  nutritive  ratio  for  all  the  rations  at 


Effect  of  Rations  Balanced  from  Restricted  Sources.  137 

approximately  1 :8.  While  we  recognize  that  for  economical 
and  rapid  growth  such  a ration  would  not  be  the  most  efficient, 
yet  it  answered  all  the  requirements  of  our  experiment. 

Lot  IV  received  a mixture  of  equal  parts  of  each  of  the  three 
rations.  This  was  to  serve  as  our  standard  ration.  There  is 
a popular  opinion  that  nutrients  from  many  sources  will  meet 
the  requirements  of  the  animal  better  than  those  from  limited 
sources.  It  was  in  obedience  to  this  idea  that  this  lot  was  es- 
tablished to  serve  for  all  standards  of  comparison. 

For  the  first  two  years  the  rations  used  were  made  up  of 
the  proportions  of  concentrates  and  roughage  shown  in  Table  I. 
For  their  formulation  the  composition  of  the  materials  was 
taken  from  the  analyses  given  by  Henry  in  “ Feeds  and  Feeding.” 
The  proportions  of  inorganic  substances  in  the  feeds  consumed 
with  a daily  intake  of  14  pounds  of  air  dry  material,  are  taken 
from  our  own  analyses.  It  was  necessary  to  restore  the  feed 
supply  about  once  a year,  involving,  of  course,  possible  but 
slight  variations  in  the  composition  of  the  materials.  But  they 
were  always  procured  from  the  same  sources,  thus  lessening 
these  variations  as  far  as  practicable. 

Where  metabolism  work  was  involved  tjie  exact  composition  of 
the  intake  was,  of  course,  determined.  The  composition  of  the 
materials  is  shown  in  Table  I. 

Lot  IV  received  a ration  made  up  of  exactly  one-third  of 
each  of  the  three  other  rations.  The  production  or  net  available 
energy  value  of  the  rations  was  calculated  from  the  data  given  by 
Armsby  7.  Where  no  figures  expressing  this  value  for  a partic- 
ular feed  used  were  available,  it  was  assumed  to  have  the  same 
value  as  the  grain  from  which  it  was  derived.  Wheat  gluten, 
for  example,  was  given  the  same  value  as  wheat  meal,  and  oat 
meal  the  same  value  as  corn  meal.  This  must  be  approximately 
true  since  almost  all  of  the  fiber  has  been  removed  in  the 
preparation  of  the  oat  meal. 

That  phase  of  the  experiment  extending  over  the  first  two 
years  and  involving  the  use  of  a ration  based  upon  equal  pro- 
portions of  digestible  nutrients  will  be  discussed  first.  After 
the  first  two  years  of  feeding,  the  rations  were  so  adjusted  as 
to  provide  equal  amounts  of  production  therms  according  to 
the  tables  of  Kellner  and  Armsby,  and  that  period  of  the  ex- 
periment will  receive  separate  treatment. 


7 Farmers’  Bui.  346,  p.  15,  U.  S.  Dept,  of  Agr. 


138 


Wisconsin  Experiment  Station. 


As  our  animals  had  done  welEon  the  corn  ration,  we  used 
that  ration  as  the  standard  for  nutritive  ratio  and  production 
therms  in  our  readjustment.  It  had  a nutritive  ratio  of  approx- 
imately 1 :8.  and  with  a consumption  of  14  pounds,  contained 
7.8  production  therms,  according  to  the  tables  of  Armsby.  The 
oat  and  wheat  rations  were  adjusted  to  that  standard  by  cut- 
ting down  the  intake  of  straw  and  increasing  the  grain  some- 


Table  I Composition  of  Feed  Given  Each  Lot  and  the  Proportion 

of  Nutrients 


Air  dry 
material 
lbs. 

Digestible  nutrients 

Nutritive 

ratio 

Pro- 

duction 

energy 

value 

therms 

Protein 

lbs. 

Carb.  Fats 
lbs.  lbs. 

Lot  I 

Corn  meal 

5 

2 

7 

.40 

.41 

.12 

3.33 

.97 

2.26 

.22 

.17 

.05 

Gluten  feed 

Corn  stover 

Total 

14.0 

.93 

6.56 

.44 

1:8 

7.87  ‘ 

Lot  11 

Ground  wheat 

6.7 

0.3 

7.0 

.68 

.21 

.03 

4.53 
.03 

2.54 

.11 

.003 

.03 

Wheat  gluten 

Wheat  straw 

Total 

14.0 

.92 

7.10 

.143 

1:8 

6.94 

Lot  III 

Oat  meal '. 

7.0 

7.0 

.80 

.08 

3.64 

2.70 

.36 

.05 

Oat  straw 

Total . 

14.0 

.88 

6.34 

.41 

1:8 

7.69 

Lot  IV 

Mixture 

[ 

Total 

1:8 

7.48 

Inorganic  Constituents  in  Air-Dried  Material 


Airdry 
mater- 
ial lbs. 

CaO 

grms. 

MgO 

grms. 

KsO 

grms. 

NagO 

grms. 

p8o5 

grms. 

SO3 

grms. 

Cl 

grms. 

Si02 

grms. 

Lot  I 

Corn  meal 

5 

.77 

4.69 

11.80 

.68 

14.75 

7.97 

1.02 

1.13 

Gluten  feed  . . . 

2 

3.80 

4.73 

3.16 

.26 

12.25 

12.62 

1.52 

0.46 

Corn  stover 

7 

23.45 

23.10 

60.06 

2.86 

15.89 

9.21 

2.63 

149.90 

Total 

14.0 

28.02 

32.52 

75.02 

3.80 

42.89 

29.80 

5.17 

151.49 

Lot  II 

Ground  wheat. . 

6.7 

2.22 

7.20 

13.9 

1.21 

27.98 

12.77 

1.33 

2.09 

Wheat  gluten.. 

0.3 

.17 

.09 

1.3 

.03 

.68 

2.85 

0.13 

.07 

Wheat  straw 

7.0 

14.61 

7.72 

21.61 

2.54 

11.39 

11.12 

4.13 

219.6 

Total 

14.0 

17.00 

15.01 

36.81 

3.78 

40.05 

26.74 

5.59 

221.76 

Lot  Ilf 

— 

TT'— ~ 

Oat  meal 

7.0 

1.74 

6.86 

11.43 

1.55 

31.78 

17.47 

1.27 

1.90 

Oat  straw 

7.0 

26.88 

17.98 

58.4 

5.7 

15.80 

15.47 

3.81 

75.63 

Total 

I 

-14.0 

28.62 

24.84 

69.83 

7.25 

47.58 

32.94 

5.08 

77.53 

Effect  of  Rations  Balanced  from  Restricted  Sources.  139 


what.  The  proportions  of  air  dried  material  calculated  to 
provide  7.8  therms  with  a consumption  of  14  pounds,  are  given 
in  Table  II. 

The  animals  were  fed  the  rations  dry.  The  straws  were  cut 
and  the  corn  stover  shredded ; the  grains  were  fed  as  meals.  The 
animals  were  fed  twice  daily,  being  given  such  portions  of  each 
mixture  as  they  would  entirely  consume.  Accurate  records  of 
consumption  were  kept.  No  attempt  was  made  to  feed  all  the 
individuals  the  same  quantity,  but  each  according  to  its  own 
demands.  Water  and  salt  were  given  adlibitum. 


Table  II  Proportions  op  Air-Dried  Material  Providing  7.8 

Therms 


Feed 

Pounds 

Nutritive 

ratio 

Production 

therms 

Corn  meal 

5.0 

Gluten  feed  

2.0 

Corn  stover 

7.0 

Total 

14.0 

1:8.2 

7.8 

Oat  meal 

8.25 

Oat  straw 

5.75 

Total 

14.0 

1:7.8 

7.8 

Wheat  meal 

8.0 

Wheat  gluten 

.3 

Wheat  straw 

5.7 

Total 

14.0 

,:7.6 

7.8 

Much  credit  is  due  William  Yoss  for  his  great  care  and  ac- 
curacy in  keeping  the  records  and  for  the  successful  handling 
of  the  feeding  side  of  the  experiment. 

The  animals  were  kept  in  the  fairly  well  lighted  basement  of 
the  University  dairy  barn  and  allowed  outdoor  exercise  in  a 
vegetation-free  paddock  during  all  days  that  the  wTeather  wtould 
allow. 

Rates  of  Growth  of  the  Animals 

The  experiment  was  started  May  31,  1907.  After  the  first 
two  years  the  rations  were  so  adjusted  as  to  provide  equal 
amounts  of  production  therms  according  to  the  tables  of  Kellner 
and  Armsby.  The  animals  wTere  weighed  monthly  and  photo- 
graphed every  six  months.  We  fully  recognize  the  inaccur- 
acies involved  in  estimating  the  progress  of  actual  tissue  build- 
ing  by  gains  in  gross  live  weight.  This  is  particularly  true 


140 


Wisconsin  Experiment  Station. 


where  the  experiment  is  continued  over  short  periods  of  time, 
as  three  or  four  months,  and  may  not  entirely  disappear  when 
continued  over  longer  periods.  But  where  the  period  is  as  long 
as  here  recorded  we  believe  such  inaccuracies  must  almost 
wholly,  if  not  entirely,  disappear. 

In  Table  III  are  given  the  annual  gains  of  each  individual, 
her  average  consumption  of  food,  water  and  salt,  and  her  daily 
yield  of  milk  during  a period  of  thirty  days  after  the  birth  of 
each  calf. 

The  rates  of  growth  of  the  animals  on  the  various  rations 
were  very  similar.  At  the  end  of  the  first  year  the  highest  in- 
dividual gain  was  in  the  corn  lot  and  the  lowest  in  the  wheat 
lot.  On  the  other  hand  there  were  individuals  in  the  wheat  lot, 
especially  No.  561,  which  equaled  and  even  exceeded  the  rate  of 
growth  of  certain  individuals  in  the  corn  lot.  The  comparison 
between  these  two  lots  is  made  because  it  will  ultimately  be 
shown  that  the  greatest  differences  in  respect  to  vigor,  resistance 
and  general  metabolism,  lay  between  them.  The  other  two  lots 
of  animals  stood  between  these  two  extremes  in  rates  of  growth. 
The  rate  of  growth  on  the  mixture  of  ingredients  was  no  better 
than  when  the  sources  were  limited. 

Of  course  the  individual  components  of  the  nutrients,  even 
from  a single  plant,  are  many ; the  principal  proteins  in  cereal 
grains  involved  in  this  experiment  are  known  to  differ  in  the 
kind  and  proportion  of  amino  g.cids  they  contain.  When,  how- 
ever, the  total  protein  of  the  grains  is  considered,  it  may  be 
found  that  the  variation  in  amino-acid  content  will  not  be  so 
great  as  when  the  individual  proteins  are  considered.  Such 
data  are  today  unavailable. 

So  far  as  the  total  proteins  of  the  rations  are  concerned,  they 
did  not  appear  to  influence  in  the  least  the  rates  of  growth  when 
their  source  was  limited  to  but  a single  plant.  Had  the  source 
of  the  proteins  been  an  important  factor  in  determining  the 
rate  of  growth,  then  the  animals  fed  the  mixture,  other  things 
being  equal,  should  have  advanced  more  rapidly.  This  is  to 
assume  that  the  animals  on  limited  sources  of  proteins  have  no 
power  of  synthesizing  a missing  amino  acid  needed  for  the  con- 
struction of  tissue  protein.  This,  however,  is  an  assumption  for 
which  at  present  there  are  limited  data  and  which  needs  further 
investigation,  at  least  for  some  of  the  amino  acids. 


Effect  of  Rations  Balanced  from  Restricted  Sources.  141 


Table  III  Initial  Weight,  Annual  Gain,  Feed  Consumed,  and  Milk 
Pkoduced  During  Period  of  Thirty  Days  After  Birth 
of  Each  Calf 


Ration 

fed 

No. 

of 

ani- 

mal 

Year 

Lbs.  initia 
weight 
and  an- 
nual gain 

Lbs.  feed 
consumed 
per  day 

Lbs.  water 
consumed 
per  day 

* Lbs.  salt 
consumed 
per  year 

Pounds  milk 
per  day. 

(30  day  period) 

Corn 

556 

1907 

413 

Corn 

Corn 

Corn 

1908 

1909 

1910 

566 

15.19 

28.4 

47.5 

123 

37 

16.36 

16.79 

40.6 

50.5 

38.5 

64.0 

26.08 
24.  JO 

Corn 

558 

1907 

344 

Corn 

Corn 

Corn 

1908 

1909 

1910 

452 

168 

164 

15.30 

17.80 

33.1 

45.7 

57.6 

20.5 

27.0 

25.0 

22.02 

31.00 

Corn 

566 

1907 

335 

Corn 

1908 

1909 

1910 

422 

12.43 

26.5 

52.5 

Corn. .... 
Corn 

104 

126 

14.32 

16.75 

41.5 

57.1 

63.0 

67.5 

27.25 

29.90 

Corn 

572 

1907 

338 

Corn 

1908 

1909 

1910 

445 

12.92 

30.9 

30.0 

Corn 

125 

174 

15.46 

16.79 

45.4 

51.1 

35.5 

29.0 

20.80 

26.70 

Oats 

557 

1907 

397 

Oats 

C nf  g 

1908 

1909 

1910 

481 

14.18 

37.5 

10.0 

a IS 

Oats 

111 

160 

16.21 

18.64 

52.4 

66.2 

48.0 

43.5 

21.12 

32.30 

Oats 

567 

1907 

347 

Oats 

On  f C 

1908 

1909 

1910 

415 

181 

150 

12.41 

39.2 

4.5 

Gdlo 

Oats 

14.88 

17.87 

45.9 

58.3 

6.5 

7.5 

22.63 

27.90 

Oats 

568 

1907 

367 

Oats 

1908 

1909 

1910 

384 

11.96 

38  6 

6.5 

Uatb 

Oats 

96 

390 

14.19 

17.08 

50.0 

54.8 

9.5 

5.5 

13.68 

Killed  Mar.  26. 

Oats.  ... 

569 

1907 

310 

Oats 

vlafo 

1908 

1909 

1910 

351 

11.16 

32.3 

8.5 

W3/t»S 

Oats 

198 

379 

14.66 

16.44 

45.6 
45.0  1 

22.5 

22.5 

20.00 

Killed  Mar.  17. 

Wheat.. . 

561 

1907 

413 

Wheat.. . 

V\/  hoot 

1908 

1909 

1910 

427 

14.47 

41.9 

33.50 

vv  near. . . 
Wheat.. . 

11 

136 

15.43 

15.68 

62.6 

60.2 

50.00 

15.50 

5.30 

Died  anthrax 
Oct.  28,  1909. 

Wheat. . . 

565 

1907 

348 

W'heat. . . 
Wheat. . . 
Wheat... 

1908 

1909 

1910 

359 

12.86 

37.4 

32.0 

86 

220 

15.53 

17.76 

53.4 

53.3 

50.5 

22.0 

13.29 

Found  dead, 
Jan.  3,  1910. 

Wheat.. . 

570 

1907 

311 

Wheat. . . 
Wheat... 
Wheat. . . 

1908 

1909 

1910 

265 

9.87 

36.0 

21.5 

131 

183 

14.06 

15.93 

50.5 

53.6 

34.0 

21.5 

10.91 

16.70 

Wheat. . . 

571 

1907 

347 

Wheat. . . 
Wheat. . . 
Wheat 

1908 

. 362 

12.19 

32.0 

26.5 

1909 

1910 

150 

240 

14  55 
17.13 

50.1 
HO. 2 

46.0 
42  5 

2.68 

15.60 

142 


Wisconsin  Experiment  Station. 


Table  III  ~ Continued  Initial  Weight,  Annual  . Gain,  Feed  Con- 

- SUMED,  AND  MlLK  PlIODUCED  DURING'  PERIOD  OF  THIRTY 

Days  After  Birth  of 'E  ach  Calf 


Ration 

fed 

No. 

of 

ani-  ’ 
mal 

Year 

Lbs.  initial 
weight 
and  an- 
nual gain 

Lbs.  feed 
consumed 
per  day 

Lbs;  water 
consumed 
per  day 

Lbs.  salt 
consumed 
per  year 

Pounds  milk 
per  day. 

(30  day  period) 

Mixture . 

554 

1907 

352 

Mixture . 

-1908 

361 

12.84- 

-37.2  - 

127.  ~ 

Mixture. 

1909 

241 

14.24 

= 43.3 

102. 

Aborted. 

Mixture. 

1910 

223 

14.93  • 

43.6 

57.5 

Killed  Mar.  7. 

Mixture . 

555 

1907 

413 

Mixture. 

1908 

455 

14.68 

: 39.7 

140. 

Mixture. 

1909 

—14 

16.28< 

52.0 

91.5 

19.32 

Mixture. 

■1910 

435 

18.94 

59.5 

89.5 

Not  pregnant. 

Mixture 

562 

1907 

340 

Mixture. 

1908 

405 

12.62 

. 28.4 

58.0 

Mixture. 

1909 

156 

14.63 

' 42.5 

37.5 

18.44 

Mixture. 

1910 

64 

16.09 

; 47.i 

■ 25.5 

- 21.00 

Mixture . 

563  i 

1907 

322 

Mixture . 

1908 

j 419 

; 12  45  ■ 

1 27  7 

i 52  0 

Mixture. 

1909 

• 110 

I i-  14 1 30 

i 41.3 

* 39.5  ; 

21.72 

Mixture. 

' j 

1910 

41 

15.11 

l i 

. 42.2 

1 40.5 

21 ’.60  ■ 

The  "greatest  growth  in  all  cases  was  made  during  the  first 
year— May  1907  to  May  1908. ' The  grjowth  of  the  seyeral  groups 
during  the  two  subsequent  years  and  . up  to  May  1910  was  sim- 
ilar, but  in  all  cases  save  one,  constant  and  positive.  No.  555 
in  the  oat  lot  averaged  a slight  loss  during  the  year  1908 — 1909. 
In  1909 — 1910  she  made  more  rapid  growth  and  at  the  end  of 
the  three  years  was  of  a weight  comparable  with  the  rest  of  the 
animals. 

So  far  as  the  rates  of  growth  and  gains  in  live  weight  of  all 
these  animals  are  concerned  there  was  nothing  to  indicate  that 
one  ration  was  very  much  superior  to  another.  Had  these  ani- 
mals been  males  with  none  of  the  additional  strains  of  milk  pro- 
duction and  pregnancy  added  to  their  function  of  growth,  the 
evidence  from  the  records  would  reveal  little  difference  in  the 
physiological  effectiveness  of  these  rations. 

* 

Amount  of  Feed  Consumed 

During  all  the  time  of  the  experiment  there  was  no  difficulty 
in  securing  a proper  consumption  of  the  rations.  Although 
given  dry,  at  all  times  the  animals  ate  the  feed,  for  the  most 
part,  with  a good  appetite  and  relish.  Occasionally  an  animal 
went  “off  feed,”  but  such  a condition  was  as  likely  to  occur  in 
ppe  group  as  in  another.  There  was  no  abnormal  craving  in 


Effect  of  Rations  Balanced  from  Restricted  Sources.  143 

their  appetites,  although  some  of  the  animals  occasionally  ate 
small  quantities  of  the  pine  shavings  used  for  bedding.  This 
was  especially  noticeable  after  they  were  turned  back  to  their 
stalls  from  their  out-door  paddock.  Individuals  in  all  the  lots 
were  offenders  in  this  regard  and  it  was  not  a distinct  group 
characteristic. 

The  fact  that  no  difficulty  was  experienced  in  obtaining  a good 
consumption  of  these  dry  rations  must  emphasize  the  possibility 
of  appeasing  the  appetite  of  our  young  animals,  not  addicted  to 
the  habit  of  foods  of  high  flavor  and  relish,  with  those  of  a less 
savory  nature.  Difficult  experimental  rations  can  pften  be 
handled  if  the  animal  to  be  used  is  young  and  has  not  become 
accustomed  to  the  delicacies  of  the  farm  commissary. 


Table  IV  Average  Daily  Amounts  of  Air- Dried  Feed  Consumed 
by  Individuals  in  Each  Lot 


Date. 

Corn 

lbs. 

Oats 

lbs. 

Mixture 

lbs. 

Wheat 

lbs. 

1907-1908 

13.4 

12.4 

13.1 

12.3 

1908—1909 

15.3 

14.9 

14.8 

14.9 

1909—1910 

17.0 

17.5 

16.2 

16.2 

Table  IV  shows  the  average  daily  amounts  of  air-dried  feed 
consumed  by  individuals  in  each  lot.  It  was  surprising  to  find 
such  little  variation  in  the  amounts  of  feed  consumed  by  the 
several  lots. 


Digestibility  of  the  Rations 

The  total  consumption  of  feed  gave  no  indication  of  the  ef- 
fectiveness with  which  the  nutrients  were  being  removed  from 
the  tract.  Consequently,  through  three  succeeding  years  two 
individuals  from  each  lot  were  simultaneously  placed  in  the 
metabolism  stalls  and  the  digestion  co-efficients  of  dry  matter 
and  nitrogen  obtained.  The  period  for  collection  of  the  feces 
and  urine  was  seven  days  with  but  one  preliminary  day  to 
accustom  the  animals  to  their  somewhat  new  environment.  A 
longer  time  than  this  was  unnecessary  because  there  was  no 
change  in  the  nature  of  the  ration.  The  same  animals  were 
used  in  the  succeeding  years  and  the  average  of  the  two  records 
is  given  in  Table  V. 


144 


Wisconsin  Experiment  Station. 


Table  V Digestibility  of  Dry  Matter  and  Nitrogen 
Average  of  Seven  Day  Records  of  Two  Animals  from  Each  Lot 


Year. 

Per  cent 
corn . 

Per  cent 
1 oats. 

Per  cent 
wheat. 

— 1 f 

Per  cent 
mixture 

Dry  Matter: 

1908 

6G.52 
70.81 
t5t5  1 1 

66.15 
62.26 
68.83 

65.63 

65.73 

75.15 

60.04 

1909 

60.96 

1910 

DP . DO 

61.79 

Nitrogen : 

1908 

54.60 
65.54 
59  35 

Of  .Od 

ft/f  Q/f 

68.60 

1909 

04 . o4r 

CD  *)Q 

58.89 

1910 

DP . 60 

ftQ  QX 

62 . 1 7 

Do . OD 

68.05 

The  figures  indicate  that  there  was  little  difference  in  the 
thoroughness  with  which  the  different  groups  were  appropriating 
their  nutrients.  The  only  distinct  variation  was  the  higher 
co-efficient  for  nitrogen  absorption  by  the  wheat  lot  in  1910. 

Appearance  of  the  Animals 

It  was  in  the  appearance  of  the  animals  that  we  had  the  first 
positive  indication  that  the  rations  were  not  alike  in  their  effects. 
Photographs  of  each  lot  were  taken  every  six  months,  but  only 
those  taken  at  the  beginning  of  the  experiment  and  at  the  end 
of  every  3^ear  thereafter  are  here  reproduced.  (See  Figures  1 to 
S.)  A casual  glance  at  these  reproductions  will  not  convey 
to  the  eye  any  essential  differences  in  the  appearance  of  the 
several  lots.  Closer  study,  however,  of  the  pictures  of  the  in- 
dividual groups  taken  two  years  after  the-  beginning  of  the  ex- 
periment must  clearly  show  a distinct  difference  between  the 
corn  and  wheat  lots,  with  the  other  two  lots  ranking  intermediate 
in  general  appearance.  The  corn-fed  animals  looked  smooth  of  j 
coat,  fuller  through  the  barrel ; and  as  expressed  by  experienced^ 
feeders  and  judges  of  domestic  animals,  they  were  in  a better 
state  of  nutrition.  On  the  other  extreme  stood  the  wheat-fed 
group  with  rough  coats,  gaunt  and  thin  in  appearance,  small  of 
girth  and  barrel,  and  to  the  practiced  eye,  in  rather  a.  lower 
state  of  nutrition.  Yet  it  must  be  remembered  that  the  total 
weight  of  the  animals  in  the  different  lots  was  essentially  similar. 
The  wheat-fed  animals  reminded  one  of  the  straw-stack- wintered 
stock  of  many  farmers. 

The  differences  in  appearance  at  the  end  of  the  first  experi- 
mental year  and  covering  a period  involving  growth  only,  were 


Effect  of  Rations  Balanced  from  Restricted  Sources.  145 

not  so  pronounced,  although  the  corn  fed  lot  was  somewhat 
sleeker  and  smoother  than  the  others. 

The  first  calves  were  born  to  these  animals  in  the  spring  of 
1909,  or  approximately  two  years  after  beginning  of  the  experi- 
ment. The  photographs  for  May,  1909 — a date  subsequent  to 
the  period  of  calving — show  a more  pronounced  differentiation 
between  the  various  lots.  This  emphasizes  the  fact  that  mere' 
growth  as  compared  with  growth,  plus  the  strains  of  reproduc- 
tion and  milk  secretion,  was  not  so  much  affected  by  the  different 
rations  as  to  become  retro-active  on  the  appearance  of  the 
animals. 

Between  the  two  extremes  of  the  corn  and  wheat  rations 
stood  the  mixture-fed  and  oat-fed  lots,  although  some  of  the 
individuals  in  both  of  these  groups  approximated  the  best  ap- 
pearing animals  of  the  corn  lot,  while  one  individual,  No.  555, 
in  the  mixture  lot,  and  two  individuals,  Nos.  568  and  569  in  the 
cat  group  were  quite  as  poor  in  general  appearance  as  the  wheat- 
fed  animals.  Compared,  however,  as  groups,  the ’order  of  excel- 
lence in  appearance  would  be : Corn,  mixture,  oat,  and  wheat. 

The  presumption  that  the  mixture  ration  was  to  be  the  standard 
did  not  hold  true. 

In  the  wheat  lot  a peculiar  habit  developed  after  five  to  six 
month’s  feeding,  the  significance  of  which  is  not  understood. 
Continually  in  the  yard  or  stall  these  animals  would  extend  the 
tongue,  wagging  it  from  side  to  side  as  if  suffering  from  some 
local  irritation.  There  was  no  effort  to  reach  any  particular 
point  and  examination  showed  no  inflamed  parts.  At  other 
times  they  would  merely  open  the  mouth  and  roll  and  unroll  the 
tongue  in  rapid  succession  for  a minute  or  two.  This  perform- 
ance was  intermittent,  being  repeated  after  a lapse  of  a few 
minutes  or  sometimes  after  a few  hours.  No  other  group  of 
individuals  showed  this  peculiar  habit. 

Amount  of  Salt  Consumed 

It  may  be  of  interest  to  have  the  attention  called  to  the  differ- 
ences in  the  amounts  of  salt  consumed  by  the  various  lots.  Salt 
was  given  the  individuals  ad  libitum  and  accurate  records  kept 
of  the  amount  consumed.  The  most  notable  result  was  the  smal- 
ler consumption  of  salt  by  the  oat-fed  animals,  which  used  but 
291/2  pounds  the  first  year,  while  the  mixture-fed  lot  consumed  a 
total  of  377  pounds  in  the  same  period;  the  wheat  and  corn 


146 


Wisconsin  Experiment  Station. 


558  555 

Figure  1 Corn-fed  group  photographed  at  beginning  of  each  experimental  year,  May  31,  1907,  and  May  31, 


Effect  of  Rations  Balanced  from  Restricted  Sources.  147 


148 


Wisconsin  Experiment  Station, 





561  565  . 571  _ _ 

Figure  3 Wheat-fed  group,  photographed  at  beginning  of  each  experimental  year,  May  31,  190/,  ana  May  31, 


Effect  of  Rations  Balanced  from  Restricted  Sources.  149 


ISO 


Wisconsin  Experiment  Station. 


■figure  5 Oat-fed  group,  photographed  at  beginning  of  each  experimental  year,  May  31,  1S07,  and  May  31,  ISO'S. 


Effect  of  Rations  Balanced  from  Restricted  Sources.  151 


OO  >> 


152 


Wisconsin  Experiment  Station. 


Figure  7 Mixture-fed  group,  photographed  at  beginning  of  each  experimental  year,  May  31,  1907,  and  May  31,  1908.' 


Effect  of  Rations  Balanced  from  Restricted  Sources.  153 


Figure  8 Mixture-fed  group,  photographed  at  beginning  of  each  experimental  year,  May  31,  1909,  and  May  31,  1910. 


154 


Wisconsin  Experiment  Station. 


lots  consumed  1431/o  and  150%  pounds  respectively.  After  the 
first  year,  which  corresponds  to  the  time  when  the  greatest 
growth  was  made  by  all  animals,  there  was  a tendency  for  a more 
uniform  consumption  of  salt  by  all  the  lots.  However,  consider- 
ed individually,  two  of  the  oat-fed  heifers  still  continued  to 
consume  small  quantities  of  salt  during  their  entire  experimental 
period  of  oat  feeding. 

One  can  find  little  support  from  these  data  for  Bunge’s  theory 
setting  forth  the  reasons  why  herbivora  must  consume  salt.  This 
theory  supposes  that  a high  potassium  intake  with  a vegetable 
diet  displaces  some  of  the  normal  sodium  chloride  of  the  blood, 
necessitating  an  extra  intake  of  the  latter  material  to  meet  ,such 
losses. 

The  potassium  intake  of  the  wheat  ration  was  only  about  one- 
half  that  of  the  oat  ration,  yet  the  sodium  chloride  consumed  by 
the  wheat  animals  was  three  times  that  consumed  by  the  oat-fed 
animals.  It  is  true  that  the  sodium  intake  of  the  oat  ration,  ex- 
clusive of  added  sodium  chloride,  was  somewhat  greater  than 
that  in  any  of  the  other  rations,  amounting  to  7.2  grams  ofNa20 
when  fourteen  pounds  were  fed,  while  the  intake  in  each  of  the 
other  two  simple  rations  was  about  3%  grams  and  about  5) 
grams  in  the  mixed  ration.  This  small  difference  of  intake/ 
normal  to  the  ration,  could  not  possibly  account  for  an  average 
daily  individual  intake  of  but  9 grams  of  salt  by  the  oat-fed  lot 
and  some  117  grams  by  the  mixture-fed  animals.  It  will  be  re- 
membered that  the  corn  and  wheat  animals  consumed  consider- 
ably less  Salt  than  the  mixtures,  but  about  five  times  as  much  as 
the  oat  lot. 

During  the  year  1909 — 1910  because  of  the  acid  conditions  of 
the  urine  of  the  wheat-fed  animals,  a mixture  of  alkaline  salts, 
in  which  there  were  32.0  grams  of  K20,  was  given  daily  with  the 
ration  to  animals  No.  565  and  No.  571.  A record  of  intake  of 
K20  and  Na20  for  these  two  animals  is  given  in  Table  YI. 


Table  VI  Record  op  Intake  of  K20  and  Na20  For  Animals 

No.  565  and  No.  571 


/ 

> 

Year. 

Animal. 

Daily  intake 
KgO  grams.  I 

Daily  intake 
NaaO  grams. 

1908  1909 

505 

40.7 

37.3 

1909-1910 

505 

73.8 

28.3 

1908-1909 

571 

38.1 

34.2 

1909-1910 

571 

76.9 

38.0 

Effect  of  Rations  Balanced  from  Restricted  Sources.  155 

With  the  higher  intake  of  potassium,  administered  as  a car- 
v bcnate,  there  was  not  an  increase  in  the  sodium  chloride  con- 
sumption. Nearly  double  the  potassium  intake  had  no  influence 
whatever  on  the  sodium  chloride  intake. 

From  a consideration  of  all  the  facts,  there  is  no  indication 
that  the  sodium  chloride  consumption  was  regulated,  or  in- 
fluenced, by  the  amounts  of  potassium  in  the  feed  consumed. 
The  positive  needs  for  common  salt  by  this  class  of  farm  animals 
have  already  been  demonstrated,8  but  no  comprehensive  theory 
explaining  the  reason  for  these  needs  in  such  large  quantities, 
and  which  is  consistent  with  all  the  facts,  is  at  present  available. 
Evidently  this  animal  has  ways  of  keeping  the  sodium  and  po- 
tassium content  of  the  blood  constant  in  spite  of  large  variations 
in  the  food  supply. 

Oestrum  Periods 

A record  of  the  time  of  occurrence  of  the  first  and  all  sub- 
sequent oestrum  periods  of  these  animals  was  kept.  This  was 
carefully  watched  for  by  Mr.  Yoss,  the  attendant.  Just  how 
much  influence  the  state  of  nutrition* of  an  animal  may  have 
upon  this  physiological  function  is  unknown,  but  it  is  generally 
believed  by  medical  authorities  that  the  influence  is  considerable. 
While  the  rations,  especially  during  the  early  part  of  the  in- 
vestigation, were  not  believed  to  be  at  all  different  in  their  nu- 
tritive value,  nevertheless  all  observations  that  could  be  brought 
to  bear  on  the  problem  wTere  made.  In  studying  the  occurrence 
of  the  first  oestrum  periods  it  must  be  remembered  that  the 
exact  age  of  the  animals  when  purchased  was  unknown,  yet  a 
variation  in  their  ages  of  from  four  to  eight  weeks  was  probably 
the  maximum.  The  dates  in  the  first  section  of  Table  VII  in- 
dicate the  occurrence  of  the  first  oestrum  with  each  individual. 

All  the  animals  were  finally  bred  to  the'  pure  bred  Guernsey 
bull,  Coralette’s  Son,  at  known  dates  during  the  period  from 
May  to  August,  1908.  There  was  no  difficulty  in  getting  the 
animals  to  breed,  although  it  was  necessary  to  re-serve  several 
of  the  individuals  at  re-occurring  oestrums.  No  one  particular 
lot  was  more  troublesome  in  this  regard  than  any  other.  Table 
VII  shows  dates  of  oestrum  periods. 


8 S.  M.  Babcock,  22nd  Ann.  Rept.,  1905,  Wis.  Exp.  Sta.,  p.  129. 


156 


Wisconsin  Experiment  Station. 


Table  YII  Dates  of  Oestrum  Periods  of  the  Individuals 


No.  of 
animal. 

Oes- 

trum 

1907. 

Oes- 

trum 

1908. 

Calv- 

ing 

1908. 

Calv- 

ing 

1909. 

Oes- 

trum 

1908. 

Oes- 

trum 

1909. 

Calv- 

ing 

1910. 

Oes- 

trum 

1910. 

Corn 

—15 

5—8 

6-2 

5—7 

4—24 

7-6 

5—7 

558 

3 — 12 

3—25 

3—29 

566 

10—18 

4—13  . 

5—7 

572 

12—18 

3—26 

4—20 

4—9 

6 — 8 

Wheat 

561 

2—28 

2—27 

7—17 

565 

10-25 

3—16 

6—3 

570 

5 — 6 

2—26 

6—1 

2—12 

4—14 

571 

4—7 

2—12 

5—22 

3—3 

5—29 

Oats 

557 

12—26 

3—4 

4—16 

3—14 

7—8 

567 

11—6 

- 

5—13 

6—3 

4—10 

6—9 

568 

12—18 

3—11 

5—16 

5-4 

569 

12—18 

3—24 

Mixture 
554 

8—24 

10—29 

12-9 

555 

10—15 

4—9 

8—22 

4—9 

l 8—22 

562 

9—22 

4—7 

4—26 

4—2 

| 5—11 

563 

10—5 

3-18 

4. .29 

4—21 

6—16 

One  would  be  inclined  from  the  first  part  of  the  data  in  Table 
VII  to  regard  any  positive  and  stimulating  influences  accruing 
from  the  various  rations  to  be  more  pronounced  and  persistent 
in  the  case  of  the  mixture  and  oat  lots ; but  it  is  more  than  prob- 
able that  the  rations  up  to  this  time  were  having  little  or  no 
effect  on  the  reproductive  functions  of  the  animals  and  that < 
variations  in  the  occurrence  of  the  oestrum  periods  were  to  be 
attributed  to  age  and  individual  idiosyncrasies.  This  statement  • 
is'  made  in  view  of  other  facts  relative  to  the  wheat  and  corn 
lots  to  be  developed  later. 

The  second  section  of  the  table  gives  the  data  on  the  re-occur- 
rence of  the  oestrum  periods  after  the  birth  of  the  first  calf. 
This  section  of  the  data  is  of  more  significance  than  that  given 
for  the  first  year  because  of  lack  of  knowledge  of  the  exact  age  < 
of  the  animals.  The  uniformly  shorter  period  of  time  between 
the  birth  of  the  calf  and  the  re-occurrence  of  the  oestrum  periods 
in  the  corn  lot  as  compared  with  a much  longer  period  for  the 
wheat  lot  should  be  noticed.  There  were  individuals  in  the 
other  two  lots  which  approached  either  extreme  found  among 
the  animals  fed  either  corn  or  wheat.  Four  to  six  weeks  after 
the  birth  of  the  first  calf  the  corn-fed  mothers  were  again  phy- 
siologically active  in  reference  to  their  oestrums.  In  the  wheat- 
fed  group  this  lapse  of  time  was  from  about  ten  to  eighteen 
weeks. 


Effect  of  Rations  Balanced  from  Restricted  Sources.  157 

After  the  birth  of  the  second  calf  in  1910,  there  was  less  ir- 
regularity in  this  respect  in  the  several  lots.  Just  how  far  the 
rations  were  responsible  for  the  irregularities  that  did  positively 
and  constantly  occur,  as  for  example,  in  the  period  after  the 
birth  of  the  first  calves  in  the  wheat  lot,  it  is  not  possible  to 
state.  It  seems  probable,  however,  when  these  data  are  cor- 
related with  other  evidence  to  follow,  that  the  wheat  ration  as 
a whole  tended  to  disturb  the  normal  functions  of  reproduction. 
It  was  in  this  lot  where  the  greatest  irregularities  occurred  es- 
pecially after  the  birth  of  the  first  calf. 

Size  and  Vigor  of  the  Young 

During  the  first  two  years  of  this  experiment  and  including 
the  first  period  of  gestation,  all  of  the  animals  were  receiving 
rations  supplying  equal  quantities  of  digestible  nutrients. 
Reference  to  Table  I will  show  that  these  rations  were  not  ex- 
actly alike  in  the  amounts  of  production  therms  supplied.  The 
wheat  ration  supplied  a slightly  lower  amount  than  all  the 
others,  the  corn  ration  furnishing  the  maximum  amount  of  pro- 
ductive energy.  The  differences  are  not  large,  the  wheat  ration 
with  a consumption  of  14  pounds  supplying  6.94  production 
therms,  while  the  com  ration  with  like  consumption  supplied 
7.87.  So  striking  were  the  effects  of  the  two  rations  on  the 
character  of  the  offspring,  that  during  the  second  gestation 
period,  1909  1910,  all  the  rations  were  so  modified  as  to  pro- 

vide equal  production  therms.  The  amount  supplied  by  the 
corn  ration  was  taken  as  the  standard  and  the  other  rations 
modified  by  decreasing  somewhat  their  straw  content  and  in- 
creasing the  grain  supply.  In  all  the  experiments  conducted 
with  these  animals  after  May  31,  1909,  or  with  other  experiments 
inaugurated  for  the  first  time,  the  rations  to  be  compared  were 
placed  on  an  equal  production  therm  basis. 

The  records  for  reproduction  for  the  year  1909-10  with 
both  standards,  are  given  in  Tables  VIII  and  IX. 

Some  important  faGts  are  contained  in  the  data  in  Tables  VIII 
and  IX.  The  corn-fed  mothers  invariably  carried  their  young 
to  within  a few  days  of  the  calculated  time  for  parturition.  In 
the  first  year  this  time  averaged  six  days,  while  in  the  second 
year  an  average  of  two  to  three  days  over-due  was  the  record. 
Invariably  the  wheat-fed  mothers  dropped  their  calves  at  from 
two  to  five  weeks  before  the  calculated  time.  The  oat  and  mix- 


158 


Wisconsin  Experiment  Station. 


ture  groups  were  more  like  the  com  lot  in  regard  to  the  time 
of  parturition. 

Table  VIII  Calves  Produced  in  1909.  Guernsey  Bull  Used  Was 

Coral  ette’s  Son 


No.  of  cowr 

Date 

served. 

Date 

due. 

Date 

calved. 

| 

Sex.  1 

Weight 
of  calf. 

Average 

weight. 

Days 

calved 

before 

time. 

Corn 

1908 

1909 

1909 

Lbs. 

*)5(5 

7—30 

5—11 

5—  8 

Hf 

70 

3 

558 

6—23 

4—  4 

3—25 

Hf 

69 

10 

566 

7—  7 

4—18 

4—13 

Hf 

67 

5 

572 

6-20 

4-1 

3—26 

Hf 

85 

73 

6 

Wheat 

561 

6—  3 

3—15 

2—27 

Hf 

52 

16 

565 

7—  4 

4—15 

3—16 

H i 

40 

30 

570 

6—11 

3—23 

2—26 

B 

48 

25 

571 

6-  8 

3—20 

2—12 

B 

44 

46 

34 

Oats 

557 

6—  6 

3—18 

3—  4 

B 

71 

14 

567 

8—14 

5—26 

5—13 

B 

77 

13 

568 

6—11 

3—26 

3—11 

B 

69 

12 

569 

6—23 

4—  4 

3—24 

B 

67 

71 

11 

Mixture 

554 

6—  4 

3—16 

10—26  a 

B 

7 a 

1 

555 

7—11 

4—22 

4—  9 

B 

58 

13 

562 

7—  5 

4' — 16 

4—  7 

Hf 

55 

9 

563 

6—19 

3—31 

3—18 

B 

65 

59 

13 

a Aborted  1908 

Table  IX  Calves  Produced  in  1910.  Bull  Used  was  Guernsey 

Sequel 


No.  of  cow. 

Date 

served 

1909. 

Date 

due 

1910. 

Date 

calved 

1910. 

Sex. 

Weight 

of 

calf. 

Aver- 

age 

weight. 

— f 

Days 

calved 

before 

time. 

Corn 

556 

7—10 

4—21 

4—24 

Hf 

84 

3 over 

558 

6—14 

3—26 

3—29 

Hf 

77 

3 over 

6—30 

4—11 

4—11 

Hf 

84 

0 over 

572 

6—24 

4—  5 

4—  9 

Hf 

93 

84i 

4 over 

Wheat 

561 

7—17 

4—28 

Cow  dead 

565 

7—  2 

4—13 

Cow  dead 

570 

6—  1 

3—13 

2— i2 

Hf 

47 

29 

571 

6-10 

3-22 

3—  3 

B 

57 

52 

19 

Oats 

557 

6 3 

3—15 

3—14 

II  f 

74 

1 

567 

7—  2 

4—13 

4—10 

Hf 

70 

3 

568 

6—  5 

3—17 

3—20 

B 

82 

3 over 

569 

6—12 

3—24 

3—15 

B 

73 

74J 

9 

Mixture 

554 

5—30 

3—11 

3—  1 

Hf 

64 

10 

555 

8 22 

6—  3 

562 

6—22 

4—  3 

4—  2 

I B 

67 

1 

563 

7—15 

4—26 

4-21 

Hf 

66 

65  it 

5 

Effect  of  Rations  Balanced  from  Restricted  Sources.  159 


On  the  equal  therm  basis,  prescribed  for  the  second  year  0? 
gestation,  the  same  conditions  prevailed  in  both  lots.  The  wheat 
mothers  gave  birth  to  their  young  from  three  to  four  weeks 
ahead  of  time,  while  the  corn -fed  mothers  carried  their  young 
past  the  calculated  time.  Again  the  oat  and  mixture  lots  were 
much  like  the  corn  lot  in  this  respect. 

The  weights  of  the  corn  calves  at  birth,  either  individually 
or  collectively  as  a group,  were  much  greater  than  those  of  the 
calves  from  the  wheat  fed  mothers.  The  corn  calves  averaged 
for  the  first  year,  73  pounds,  while  the  wheat  calves  averaged 
but  46  pounds.  The  calves  born  to  the  oat-fed  mothers  were 
nearly  as  large  as  those  from  the  corn-fed  group,  while  the  mix- 
dure-fed  mothers,  excluding  the  abortion  of  No.  554,  produced 
calves  averaging  59  pounds.  In  the  order  of  gross  average  weight 
the  calves  stood  as  follows  for  the  first  year:  Corn,  oats,  mix- 

ture, and  wheat..  Here  again,  contrary  to  expectation  and  to 
the  popular  belief  of  a greater  efficiency  of  a mixture  without 
prescribing  its  source,  the  mothers  on  the  corn  ration  were  per- 
forming their  normal  physiological  processes,  with  evidence  of 
greater  strength  and  vigor  than  any  of  the  other  lots. 

In  1909 — 10  on  the  equal  production  therm  basis,  the  matter 
of  size  of  offspring  was  an  exact  counterpart  of  the  record  0!’ 
the  previous  year.  The  corn  mothers  produced  calves  averaging 
84%  pounds,  while  the  two  wheat  mothers  remaining  in  that 
group,  produced  calves  averaging  52  pounds.  The  average  for 
the  other  two  groups,  oat  and  mixture,  were  74  and  65  pounds 
respectively. 

The  variations  in  the  vigor  of  the  offspring  from  the  several 
lots  were  also  pronounced.  There  are,  however,  no  scientific 
ways  of  directly  measuring  differences  in  vitality  and  unless  the 
differences  in  appearance  between  two  individuals  are  so  strik- 
ing as  to  be  immediately  noticed,  those  more  subtile  variations 
that  may  exist,  cannot  be  foretold  with  any  certainty.  In  our 
observations,  reference  is  made  to  those  large  and  easily  notice- 
able factors  such  as  ability  to  stand  and  walk  within  an  hour  or 
so  after  birth,  to  suckle  the  mother  without  aid,  etc.,  character- 
istics which  are  always  shown  by  young  vigorous  calves.  It 
is  to  be  hoped  that  science  will  some  day  furnish  us  with  an 
accurate  way  of  measuring  such  vital  phenomena  as  vigor  and 
resistance,  but  at  present  we  must  be  content  with  these  crude 


160 


Wisconsin  Experiment  Station. 


ways  6f  distinguishing  such  characteristics.  The  work  of  Hunt9 
upon  the  resistance  of  animals  fed  different  diets,  to  certain 
poisons,  is  an  effort  in  this  direction. 

The  data  in  reference  to  the  vigor  of  the  off-spring  are  so  im- 
portant to  our  subject  that  they  will  be  given  in  this  place  in 
full  for  the  years  1909  and  1910.  Attention  is  called  again  to  the 
fact  that  the  rations  of  the  several  lots  of  1909  were  alike  in  re- 
spect to  quantity  of  digestible  nutrients,  hut  derived  from  dif- 
ferent ‘sources.  In  1910  they  were  alike  in  respect  to  produc- 
tion therms. 


Corn 

No.  556 

No.  558 
No.  566 
No.  572 
Wheat 

No.  561 


No.  565 
No.  570 
No.  571 

Oats 

No.  557 

No.  567 
No.  568 

No.  569 

Mixture 
No.  554 
No.  555 


No.  562 
No.  563 


Record  of  Calves  Born  in  1909 

Calf  strong  and  vigorous ; stood  up  within  one  hour 
after  birth.  Lived. 

Calf  strong  and  vigorous.  Lived. 

Calf  strong  and  vigorous.  Lived. 

Calf  strong  and  vigorous.  Lived. 

Calf  very  weak,  could  not  stand  and  suckle  mother. 
Stood  alone  for  first  time  after  four  days.  Died  in 
convulsions  on  twelfth  day. 

Calf  weak.  Lived  two  hours. 

Calf  weak.  Lived  twelve  hours. 

Calf  bom  dead. 

Calf  weak,  did  not  get  up  for  thirty-six  hours. 
Lived. 

Calf  born  dead. 

Calf  very  weak,  was  nursed  from  bottle ; died  on 
third  day. 

Calf  weak;  could  not  get  up  alone  for  seventy-two 
hours;  nursed.  Lived. 

Abortion  due  to  accident  in  the  yard. 

Calf  fairly  strong;  got  up  after  two  hours  from 
birth,  but  could  not  walk;  was  nursed  from  bottle 
the  first  day.  Lived. 

Calf  weak;  could  not  get  up.  Lived  six  hours. 
Calf  born  dead. 


» Bui.  69,  Hygienic  Lab.  U.  S.  Treas.  Dept. 


Effect  of  Rations  Balanced  from  Restricted  Sources.  161 


Record  of  Calves  Born  in  1910 

Corn 

No.  556  Calf  strong  and  vigorous.  Lived. 

No.  558  Calf  strong  and  vigorous.  Lived. 

No.  566  Calf  strong  and  vigorous.  Lived. 

No.  572  Calf  strong  and  vigorous.  Lived. 

Wheat 

No.  561  Mother  died  of  anthrax,  October  28,  1909. 

No.  565  Mother  found  dead  in  stall,  January,  3,  1910; 

halter  strangulation  probable  cause. 

No.  570  Calf  very  weak;  could  not  get  up.  Lived  nine 
hours.  , 

No.  571  Calf  very  weak;  could  not  get  up.  Lived  twenty- 
two  hours. 

Oats 

No.  557  Calf  fairly  strong ; nursed  from  bottle  the  first  day. 
Lived. 

No.  567  Calf  fairly  strong;  did  not  get  up  for  twelve  hours; 
nursed  from  bottle.  Lived. 

No.  568  Calf  fairly  strong;  nursed  from  bottle  for  forty- 
eight  hours.  Lived. 

No.  569  Calf  not  strong  enough  to  get  up  for  twenty-four 
hours;  nursed  from  bottle.  Lived. 

Mixture 

No.  554  Calf  born  dead. 

No.  555  Not  with  calf. 

No.  562  Calf  very  weak;  with  help  stood  up  after  forty- 
eight  hours;  suckled  mother.  Lived. 

No.  563  Calf  fairly  strong;  stood  up  and  suckled  mother 
after  a few  hours.  Lived. 

In  addition  to  the  above  records  photographs  of  a representa- 
tive from  -each  lot  for  both  1909  and  1910  are  given  in  Figures 
9 to  17.  Photographs  of  the  calves  alone  are  given  for  1909. 
For  1910  reproductions  of  both  calf  and  mother  are  shown. 

The  strong,  large,  and  vigorous  type  of  calf  from  the  corn-fed 
mothers  is  a group  characteristic,  while  the  small,  weak  offspring 
from  the  wheat-fed  mothers  is  characteristic  for  that  group. 
The  other  two  groups  are  intermediate  in  respect  to  the  character 
of  the  offspring.  Considered  for  both  years,  the  calves  from  the 
oat-fed  mothers  were  superior  in  respect  to  strength  and  vigor 
to  the  calves  from  the  mixture-fed  mothers.  So  consistent  are 


162 


Wisconsin  Experiment  Station, 


Figure  9 Representative  calves  from  each  group  in  1909. 

Upper.  CoYn  calf.  Mother  572.  Third.  Oat  calf.  Mother  569. 

Second.  Wheat  calf.  Mother  661.  Lower.  Mixture  calf.  Mother  555. 


Effect  of  Rations  Balanced  from  Restricted  Sources.  1 63 

the  records  for  each  lot  that  it  appears  practically  certain  that 
the  influences  are  directly  attributable  to  the  rations  fed  and 
not  to  mere  inherent  idiosyncrasies  of  the  individual. 

Post  mortem  examinations  made  on  the  dead  wheat  calves  re- 
vealed nothing  that  would  indicate  that  these  animals  were  not 
fully  developed  and  provided  with  all  anatomical  structures; 
but  they  lacked  size  and  that  subtle  something  which  constitutes 
vigor  and  strength. 

During  the  periods  of  gestation,  as  well  as  at,  all  other  times, 
the  consumption  of  food  in  the  several  lots  was  not  unlike.  The 
same  statement  can  be  made  in  reference  to  the  absorption  of 
food,  as  measured  by  the  coefficients  of  digestibility  of  dry  mat- 
ter and  of  nitrogen.  The  results  with  the  wheat  ration  cannot 
possibly  be  attributed  to  mere  inanition,  in  the  sense  of  a lack  of 
supply  of  available  food  materials. 

These  results  emphatically  show  how  depressing  or  stimulat- 
ing the  influence  of  a ration  may  become,  even  when  it  is  made 
up  of  supposedly  normal  feed  materials  and  balanced  as  to 
ordinary  chemical  constituents  and  supply  of  energy,  especially 
when  that  ration  is  continued  for  a long  time.  The  evidence  for 
the  necessity  of  giving  much  weight  to  the  physiological  influence 
of  the  ration,  apart  from  its  digestible  protein  content  and  cal- 
orific value,  is  positive.  The  facts  already  brought  out  from 
this  investigation  should  emphasize  strongly  the  necessity  for 
further  study  with  all  classes  of  farm  animals,  as  well  as  in 
human  dietetics,  of  the  physiological  value  of  various  rations 
and  diets;  and  these  values  can  be  secured  with  certainty  only 
when  the  experimental  animal  has  been  involved  in  both  func- 
tions of  growth  and  reproduction. 

The  observations  of  Watson  and  Hunter10  on  the  influence  of 
diet  on  the  growth  and  nutrition  of  rats  illustrate  how  foods, 
with  a chemical  composition  at  least  superficially  alike,  may  re- 
act differently  in  the  animal  body.  Of  fourteen  young  rats  fed 
on  a porridge  of  boiled  oat  meal  with  water  and  skim  milk,  all 
but  two  succumbed  within  five  months;  while  those  receiving  a 
diet  of  bread  and  skim  milk  thrived  as  usual.  The  same  invest- 
igators give  another  illustration  of  the  influence  of  diet  on  the 
progeny,  which  is  quoted  by  Chittenden  in  “Nutrition  of  Man,” 
as  follows:  “Thus  of  ninety-three  rats  bom  of  meat-fed  par- 


10  Journal  of  Physiology  Vol.  34,  1906,  p.  112. 


164 


Wisconsin  Experiment  Station. 


ents,  only  nineteen  were  alive  at  the  end  of  two  months,  while 
of  ninety-seven  young,  born  of  bread-and-milk-fed  rats,  eighty 


Figure  10  Wheat  cow  570.  1010. 

two  were  alive  and  in  appar- 
ent health  at  the  end  of  the 
same  period. 

Milk  Secretion 
In  addition  to  the  observa- 
tions made  on  the  character 
of  the  calves  produced  with 
the  several  rations,  a record 
of  the  yield  and  composition 
of  the  milk  secreted  was  also 
m'ade  as  shown  in  Table  X.  This  record  extended  over  a period 
of  thirty  days  immediately  following  the  cessation  of  the  produc- 

Table  X Yield  and  Composition  of  Milk  for  Thirty  Days  After 
the  Cessation  of  Production  of  Colostrum  Milk,  1909 


Figure  11  Wheat  calf  1910.  Mother  570. 


Lot. 

Num- 
ber of 
ani- 
mals. 

Total 
yield  per 
lot,  lbs. 

Average 
individ- 
ual daily 
yield 
lbs. 

! 

Per 

cent 

solids. 

Per  cent 
total 
proteins. 

1 

Per 

cent 

casein. 

[ 

Per 

cent 

fat. 

Per 

cent 

ash. 

cc.  N /10 
alkali  re- 
quired for 
25  cc.  milk. 

Corn 

4 

2884.9 

24.03 

12.43 

3.19 

2.50 

3.45 

.66 

5.6 

Wheat. .. 

4 

965.8 

8.04 

12.30 

3.08 

2.50 

3.55 

.72 

6.6 

Oat 

4 

2326.8 

19.38 

13.03 

3.09 

2.55 

4.25 

.67 

5.9 

fixture. 

3 

1784. 

19.82 

14.10 

3.10 

2.50 

5.65 

.69 

5.7 

Effect  of  Rations  Balanced  from  Restricted  Sources.  165 


tion  of  colostrum  milk.  The  data  for  the  composition  of  the  milk 
in  1909  represent  the  average  of  separate  analyses  of  the  milk 


Figure  12  Corn  cow  566.  1610. 


composited  from  two  individ- 
uals from  each  group  over  a 
period  of  one  week.  The  col- 
umn headed  “Number  of  ani- 
mals” represents  the  number 
of  individuals  involved  in  the 
production  of  milk. 

The  record  for  1910,  shown 
in  Table  XI,  represents  a rec- 
ord of  yield  for  thirty  days. 
The  record  of  composition  is 


Figure  13  Corn  calf  1910.  Mother  566. 


the  average  of  the  analyses  of  daily  samples  from  two  individ- 
uals in  each  lot  for  three  days. 

Table  XI  Yield  and  Composition  of  Milk  for  1910 


Lot. 

No.  of 
ani- 
mals. 

Total  | Average 
yield  |“- 

iotr  1 
pounds. 

| 

Per 

cent 

solids. 

1 

Per 

cent 

total 

proteins 

Per 

cent 

casein. 

Per 

cent 

fat. 

Per 

cent 

ash. 

cc  N/ 10 
alkali  re- 
quired for 
25  cc.  milk. 

Corn 

Wheat . . 
Oat 

4 

2 

2 

2 

3375.1  1 28.0 

975.5  16.1 

1810.8  30.1 

1281.2  21.3 

12.40 

12.80 

12.52 

13.81 

2.87 

3.12 

2.75 

2.90 

2.40 

2.70 

2.30 

2.30 

3.80 

4.10 

3.90 

4.40 

.74 

.76 

.74 

.73 

6.1 

7.6 

6.7 
6.5 

Mixture. 

166 


Wisconsin  Experiment  Station. 


The  records  of  yield  show  the  same  low  capacity  for  the  pro- 
duction of  milk  by  the  wheat-fed  mothers  as  was  shown  in  their 


14  Oat  cow  567.  1910. 

record  for  calf  production. 
The  highest  individual  record 
for  daily  production  in  this 
lot  was  13.2  pounds  and  the 
lowest  2.6  pounds.  The  corn- 
fed  mothers  for  the  year  1909 
responded  to  their  ration 
more  fully  than  any  other  lot, 
the  lowest  individual  record 
being  20.8  pounds  per  day. 
As  in  all  of  our  other  observa- 
tions, the  mixture-fed  and  oat-fed  animals  were  intermediate 
to  the  corn  and  wheat  groups  in  respect  to  milk  production. 

In  the  year  1910  the  same  conditions  in  the  several  lots  again 
obtained,  except  that  the  oat-fed  mothers  were  slightly  ahead  of 
the  corn-fed  mothers  in  regard  to  milk  production.  All  lots 
showed  a considerable  improvement  in  their  capacity  for  milk 
yield,  but  the  wheat-fed  mothers  were  still  much  lower  than  all 
other  lots.  In  contrast  to  this  low  production  during  both 
years  by  the  wheat-fed  mothers,  there  stood  the  fact  that  the 
feed  consumption  at  all  times  during  the  period  when  these  re- 


Effect  of  Rations  Balanced  from  Restricted  Sources.  16? 


cords  were  taken  was  practically  alike  for  all  the  groups.  From 
13  to  16  pounds  of  the  air  dried  ration  in  1909  and  15  to  17 


Figure  16  Mixture  cow  562.  1910. 


pounds  in  1910  were  con- 
sumed in  the  wheat  and  corn 
lots  respectively  during  this 
period  of  milk  production. 

So  far  as  the  composition 
of  the  milk  is  concerned  the 
analyses  indicate  that  the 
product  was  in  every  respect 
perfectly  normal.  The  some- 
what higher  fat  yield  in  the 
milk  from  the  mixture-fed 
animals  is  to  be  attributed  to 
the  fact  that  this  lot  contained  one  animal  of  part  Jersey  and 
another  of  part  Guernsey  breeding.  This  constancy  in  the  com- 
position of  the  milk  produced  is  one  of  the  important  funda- 
mentals in  milk  secretion.  Under  wide  variations  in  the  char- 
acter and  effect  of  the  food  consumed,  the  gross  composition 
of  the  milk  that  is  secreted  is  normal  and  constant.  Pos- 
sibly one  variation  in  the  milk  constants  determined,  is  large 
and  definite  enough  to  receive  our  attention.  The  titra- 
tion factor  for  the  wheat  milks  in  both  years  is  constantly  higher 


Figure  17  Mixture  calf  1910.  Mother  562. 


168 


Wisconsin  Experiment  Station. 


than  for  all  other  milks.  This  may  be  due  to  several  causes.  A 
low  base  content  in  proportion  to  the  acid  radicals ; an  arrange- 
ment of  bases  and  acids  for  the  formation  of  acid  salts ; or  the 
presence  of  small  amounts  of  some  organic  acid  in  the  milk. 
None  of  these  suggestions  have  as  yet  been  confirmed  in  this  in- 
vestigation. 

The  fact  that  the  wheat-fed  mothers  constantly  produced 
urines  acid  to  phenolphthalein  and  litmus,  while  the  urines  of 
all  other  lots  were  acid  to  phenolphthalein  alone,  but  neutral  or 
alkaline  to  litmus,  is  in  harmony  with  this  condition  of  the  milk 
which  indicates  a tendency  for  the  secretions  as  well  as  the 
excretions  of  this  group  to  respond  differently  to  indicators  than 
those  fed  the  other  rations.  The  variations  in  the  titration  factor 
of  the  milks  for  the  various  lots  is  not  large  and  speaks  most 
forcibly  again  for  an  attempt  on  the  part  of  the  mother  to  pro- 
duce milk  normal  in  composition. 

Character  of  the  Milk  Fats  Produced 

Much  excellent  work  has  been  done  on  the  influence  of  cer- 
tain rations  and  added  oils  on  the  chemical  and  physical  con- 
stants of  butter  fat  and  on  the  physical  properties  of  the  butter 
made.  The  work  of  Lindsey  11  in  this  field  is  particularly  im- 
portant. 

The  opportunity  of  making  further  observations  of  the  in- 
fluence on  the  fats  secreted  in  the  milk  of  a restricted  ration  fed 
practically  through  the  whole  life  of  the  animal  was  taken  ad- 
vantage of.  In  1909  composites  of  milk  from  all  individuals  in 
each  lot  were  churned.  The  butter  produced  was  not  salted. 
From,  this  butter,  samples  of  fat  were  prepared  by  melting  and 
filtering,  and  the  physical  and  chemical  constants  determined.  A 
record  of  the  data  for  1909  appears  in  Table  XII. 


Table  XII  Composition  of  Milk  Fats  Produced  in  1909 


Constants 

Corn. 

Wheat. 

Oats. 

Mixture. 

Melting-  point 

Reichert  No 

Koettstorfer  No 

Hiibl  No 

Ref.  Index.  40° 

o 

30.9 

30.98 

226.1 

38.51 

1.455T 

30.0 
27  22 
222^50 
41.81 
1.4562 

O 

37.4 
28.89 
218.25 
38.03 
1 .4558 

32.7 
25.36 
217.80 
42.81 
1 . 4568 

ii  Mass.  Agr.  Exp.  Sta.,  21  Ann.  Rpt.,  1909,  p.  66. 


Effect  of  Rations  Balanced  from  Restricted  Sources.  169 

The  one  fact  which  the  data  emphasize  is  the  constancy,  in 
most  respects,  of  the  composition  and  character  of  the  fats  formed 
with  the  different  rations.  The  only  important  variant  was 
the  high  melting  point  of  the  oat  fats.  This  was  above  the  range 
of  normal  butter  fat  and  is,  in  a general  way,  to  be  correlated 
with  the  property  of  firmness  in  butter.  When  the  milk  fats 
from  the  several  groups  were  melted  and  then  allowed  to  stand 
at  70°  F.,  or  about  normal  room  temperature,  for  a few  hours, 
the  separation  of  the  solid  fats  took  place  as  shown  in  Figure 
18.  The  oat  fats  became  a solid  mass;  the  mixture  separated  a 


Figure  18  Milk  fats  190©  showing  separation  of  solid  fats  after  a few  hours  at  70°  F. 

Ftom  left  to  right  in  the  figure,  the  fats  were  taken  from  milk  of  corn-fed,  oat- 
fed,  wheat-fed,  and  mixture-fed  cows,  while  the  bottle  at  the  right  contains  fat 
from  milk  of  cows  fed  in  the  ordinary  way. 

large  proportion  of  solid  fats;  while  the  corn  and  wheat  fats 
separated  little.  This  property  of  separating  a portion  of  the 
solid  fats  of  high  molecular  weight  appears  to  correlate  directly 
with  the  melting  point.  A sample  of  butter  from  ordinary  milk  J 
and  marked  “ dairy’ ’ is  placed  in  the  picture  for  comparison. 

It  is  more  like  the  oat  fat  in  respect  to  the  proportion  of  solid, 
fats  it  contains.  In  fact  this  relation  of  the  melting  point  to 
the  proportion  of  solid  fats  separating  out  is  much  more  direct 
than  is  the  case  of  any  of  the  other  constants.  The  Hiibl  num- 
ber, expressing  as  it  does  the  proportion  of  unsaturated  fats  pre- 
sent, and  more  often  believed  to  control  the  melting  point  in  a 
mixture  of  milk  fats,  is  just  as  high  in  the  oat  fats  as  in  the 
com  fats. 


370 


Wisconsin  Experiment  Station. 


In  1910  the  observations  on  the  nature  of  the  milk  fats  were 
again  repeated,  but  this  time  the  milk  from  two  individuals  in 
each  lot  was  selected  and  the  fats  prepared  from  each.  This 
was  to  determine  whether  the  high  melting  point  of  the  oat  fats, 
as  a group,  was  a food  factor,  or  whether  it  was  possibly  in- 
fluenced by  certain  individuals  in  the  group.  The  data  cover- 
ing the  period  for  1910  are  displayed  in  Table  XIII. 


Table  XIII  Physical  and  Chemical  Constants  of  Milk  Fats 

for  1910 


No.  of  animal. 

Melting1 

point. 

Koettstorfer 

No. 

Hiibl  No. 

Reichert 

No. 

Ref.  Index 
40° 

Corn: 

572 

26.72° 

228.8 

38.51 

36.52 

1 . 4563 

558 

29.48° 

225.1 

36. 16 

35.49 

1 . 4565 

Wheat: 

570 

30.35° 

225.1 

37.78 

31.26 

1.4566 

571 

29.25° 

221.9 

27.59 

37.60 

1.4548 

Oats: 

557 

34.45° 

219.9 

36.13 

30.40 

1.4564 

567 

30.32° 

224.9 

36.06 

36.05 

1.4557 

Mixture: 

562 

26.72° 

226.3 

35.27 

36.65 

1.4557 

563 

29.48° 

213.4 

38.02 

27.06 

1.4571 

The  one  distinguishing  characteristic  of  the  milk  fats  from 
the  various  groups  in  1909 — namely,  a high  melting  point  in  the 
oat  milk  fats — is  apparently  not  so  distinctly  a group  and  food 
characteristic.  While  there  is  a tendency  in  this  direction  and 
the  data  on  the  individuals  in  1910  show  the  highest  melting 
point  fats  in  the  oat  lot,  nevertheless,  the  wheat  and  all  other  lots 
contained  individually-produced  milk  fats  with  melting  points 
equal  to  or  nearly  as  high  as  that  of  No.  567  in  the  oat  group. 
Again  the  fats  were  melted  and  allowed  to  stand  at  72°  to  75°  F. 
for  several  days  and  then  photographed.  (See  Figure  19.)  The 
proportion  of  solid  fats  separating,  stood  in  close  relation  to  the 
melting  points.  No.  557,  the  oat  fat  with  the  highest  melting 
point  showed  the  highest  proportion  of  solid  fats,  while  572,  a 
corn  fat,  showed  the  smallest  proportion. 

The  other  constants  showed  quite  as  marked  a variation  among 
the  individuals  of  the  same  group  as  among  individuals  of  dif- 
ferent groups.  In  most  respects  the  character  of  the  fats  secret- 
ed is  influenced  more  by  individual  metabolism  than  by  the 
character  of  the  food  intake.  This  is  particularly  true  when  the 
fat  content  of  the  intake  is  not  excessive,  and  the  content  only 


Effect  of  Rations  Balanced  from  Restricted  Sources.  171 


that  incident  to  normal  feeding  materials.  A surfeit  of  foreign 
fat  will  undoubtedly  influence  to  some  extent  the  character  of 
the  fats  secreted  in  the  milk,  as  has  been  shown  by  Lindsey  and 
his  associates. 

It  should  be  remembered  that  with  a food  consumption  of  14 
pounds  of  such  a feed  mixture  as  here  used,  the  digestible  fat 
of  the  rations  will  range  from  about  .14  pounds  in  the  wheat  to 
.44  pounds  in  the  com  ration,  with  a daily  production  in  the 
milk  of  the  wheat  animals  of  about  0.5  pounds  of  fat  and  nearly 
1.0  pound  in  the  daily  individual  secretion  of  the  com  lot.  Con- 
sequently, if  we  assume  that  the  fat  of  the  ration  is  directly 
transported  to  the  mammae  we  are  still  confronted  by  the  fact 


Figure  19  Milk  fats  1910,  showing  separation  of  solid  fats  after  a few  hours  at  72°  F 
n.5?ed  as  fol!ows  from  left  to  right:  Oats  557,  oats  567,  wheat  57l! 

wheat  570,  mixture  563,  mixture  562,  corn  558,  corn  572. 


that  at  least  one  half  of  the  milk  fat  must  be  synthesized  from 
material  not  fat.  So  different  are  the  fats  stored  in  other  parts 
of  the  animal  body  from  these  secreted  in  the  milk  that  it  is  not 
to  be  assumed  that  any  large  direct  transportation  of  fats  is  tak- 
ing place  under  conditions  of  normal  nutrition  and  maintenance 
of  body  weight. 

Our  data  support  the  conclusion  that  these  rations  had  little 
direct  and  positive  influence  on  the  chemical  constitution  of  the 
fats  secreted  in  the  milk.  The  variations  that  did  occur  were 
more  of  an  individual  nature.  Had  we  based  our  conclusions 
on  the  character  of  the  milk  fats  produced  by  the  groups  in 
1909  we  should  certainly  have  given  to  the  oat  ration  the  pro- 
perty of  producing  high  melting  milk  fats  containing  a large 
proportion  of  solid  fats,  and  to  the  wheat  and  com  rations  ex- 
actly opposite  properties.  That  such  did  not  obtain  in  any  pro- 


172 


Wisconsin  Experiment  Station. 


nonneed  degree  is  more  in  harmony  with  onr  data  for  1910  on 
the  individual  animals. 

Composition  of  Animal  Tissue 

The  influence  of  long  continued  feeding  of  the  same  ration  on 
the  distribution  of  nitrogen  in  the  tissues  and  the  chemical  con- 
stants of  the  fats  from  various  parts  of  the  body,  has  not,  so  far 
as  the  authors  are  aware,  been  studied  with  this  class  of  farm 
animals.  Jordan 12  studied  the  influence  of  narrow  and  wide 
rations  on  the  character  of  the  increase  in  growing  steers  and 
found  that,  within  the  limits  prescribed  by  his  ration,  no  in- 
fluence on  the  gross  constitution  of  the  increase  was  apparent. 
The  rates  of  growth  alone  were  different. 

In  this  connection  reference  should  be  made  to  the  work  of 
Mendel 13  on  the  influence  of  diet  on  the  chemical  composition  of 
the  animal  body.  Mendel  fed  mice  rations  varying  widely  in 
their  relative  content  of  proteins,  carbohydrates,  and  fats,  with- 
out modifying  in  any  degree  the  gross  composition  of  their  tis- 
sue, when  compared  with  the  tissues  of  a normally  fed  mouse. 

Numerous  studies  with  swine  fed  on  rations  of  limited  source 
and  particularly  one-sided  in  regard  to  protein  and  ash  have 
been  made.  Such  investigations  have  been  made  by  Sanborn,14 
Henry, 15  Shelton,16  Forbes,17  and  the  authors  (unpublished 
data)  with  corn  meal  as  the  sole  diet.  All  these  studies  indicate 
a somewhat  restricted  growth  of  skeleton,  musculature,  and  im- 
portant body  organs  on  such  a diet,  but  there  is  no  substantial 
evidence  that  these  three  important  classes  of  tissues  are  not 
normal  and  essentially  similar  in  chemical  composition  to  the 
same  tissues  built  by  an  animal  receiving  liberal  and  mixed 
rations.  More  bone  may  be  formed  in  one  case,  as  compared  with 
the  other,  but  the  proportions  of  calcium,  phosphorus  and  mag- 
nesium deposited  are  alike  in  all  cases.  The  proportion  of  or- 
ganic matter  to  the  inorganic  may  be  modified,  but  what  in- 
organic material  is  deposited  remains  of  constant  constitution. 

The  proportion  of  proteins  to  other  constituents  in  a given 
tissue  may  vary,  but  the  constitution  of  the  proteins  stored  is 


12  Ann.  Rept.  Me.  Exp.  Sta.,  1895,  p.  36. 

33  Bio.  Chemische  Zeitschrift  1908  11,  p.  281. 

i4  Mo.  Agr.  Col.  Bui.,  10,  14,  19. 

is  Kepts.  Wis.  Agr.  Exp.  Sta.,  1886-87-88-89. 

16  Kas.  Agr.  Exp.  Sta.,  Bui.  9. 

17  Ohio  Agr.  Exp.  Sta.,  Bui.  213. 


Effect  of  Rations  Balanced  from  Restricted  Sources.  173 

identical  and  constant  for  the  species  under  wide  differences  in 
diet.  The  proportion  of  the  reserve-  to  the  more  active  proteins 
of  the  cell  may  vary,  but  there  is  every  evidence  for  believing 
that  the  total  proteins  for  that  cell  and  tissue  are  constant  in 
composition  and  not  modified  by  ration  or  diet. 

On  the  other  hand,  the  work  of  Shutt 18  indicates  that  a diet 
of  com  meal  as  compared  with  mixed  diets  does  influence  the 
kind  of  fats  stored  by  swine,  a soft  pork  with  more  olein  be- 
ing the  result  of  the  long  continued  use  of  such  a ration. 

Apparently  from  these  and19  other  studies  this  class  of  tissue 
can  actually  suffer  modification  in  its  chemical  constitution 
under  the  influence  of  diet,  but  just  how  far  the  diet  is  effective 
even  in  this  regard  is  not  definitely  settled.  This  whole  ques- 
tion has  recently  been  opened  again  by  Abderbalden20  who  dis- 
tinguishes between  true  cell  fat  and  deposited  fat,  and  brings 
forth  evidence  that  the  former  does  not  vary  in  composition  with 
the  food  eaten. 

For  our  work,  one  of  the  animals  in  each  lot,  with  one  excep- 
tion, was  slaughtered  at  the  end  of  thirty-three  months  of  feed- 
ing. No.  565  in  the  wheat  lot  was  found  strangled  in  her  stall 
January  3,  1910,  and  her  tissues  were  used  as  samples  of  mater- 
ial for  that  group ; consequently  she  represents  a period  of  feed- 
ing of  thirty  months.  Various  organs,  the  blood,  and  the  lean 
tissue  of  the  thigh,  were  saved.  They  were  preserved  by  drying 
at  60°  to  70°C. 

Nitrogen  distribution  by  the  Hausmann-Osborne  method  was 
made  on  the  muscle  of  the  thigh  and  on  the  blood.  The  muscle 
tissue  was  prepared  for  analysis  by  drying,  washing  several 
times  with  water  to  remove  extractives  and  finally  extracted 
with  chloroform  for  twenty-four  hours,  and  again  dried.  The 
blood,  after  drying,  was  only  extracted  with  chloroform  and 
dried.  The  results  are  given  in  Table  XIV  and  are  expressed 
in  per  cents  of  the  total  nitrogen  of  the  tissue.  Data  on  the 
other  tissues  are  not  yet  available. 

The  results  are  uniform  for  these  different  tissues,  which  are 
the  product  of  the  continued  use  and  influence  of  these  rations 
for  nearly  three  years,  and  which,  so  far  as  the  grains  at  least 

18  Central  Exp.  Farm,  Ottawa,  Canada,  Bui.  38. 

^Lebedeff,  Pfliiger’s  Arch.  1883,  31-11;  Rosenfeld  Zeit.  f.  k.  M.  1898, 
36-232. 

20  Zeit.  fur  Physiol.  Chemie,  Vol.  65,  p.  330. 


174 


Wisconsin  Experiment  Station. 


are  concerned,  are  known  to  vary  in  the  nature  of  their  proteins, 
and  consequently  in  amino-acid  content.  Apparently  for  the 
protein  structures  there  is  a strong  tendency  for  maintaining 
constancy  in  ultimate  structure  independent  of  variations 
in  the  nature  of  ingested  proteins.  The  fact  is  not  new, 
having  been  made  the  subject  of  experimental  study  by  Abder- 
halden. 21 


Table  XIV  Distribution  of  Nitrogen  in  Muscle  and  Blood 


Per  cent 

Per  cent 

Per  cent 

Per  cent 

ammonia 

humus 

di- amino 

mono-amino 

N 

N 

N 

N 

Corn  cow  muscle 

6.29 

2.49 

26.15 

65.07 

Wheat  cow  muscle 

6.69 

3 94 

26.91 

62  46 

Oat  cow  muscle 

6.86 

3.35 

27!  79 

62^00 

Corn  cow  blood 

5.24 

5.22 

26.35 

63.19 

Wheat  cow  blood 

5.29 

4.91 

25.79 

64.01 

In  an  attempt  to  modify  the  structure  of  the  blood  proteins, 
Abderhalden  fed  a horse  proteins  of  an  amino  acid  content  widely 
different  from  that  of  the  blood  proteins  without  effecting  a 
change  in  their  constitution. 

The  fats  of  the  carcass  subjected  to  investigation  were  from 
the  omentum,  mesentery,  heart  and  kidney.  These  fats  were 
prepared  by  thoroughly  grinding  the  tissue  and  allowing  it  to 
stand  at  98°C.  for  a short  time.  The  liquid  fat  was  then  filtered 
warm.  In  the  case  of  the  heart  the  fat  represents  not  only 
that  immediately  extraneous  to  the  organ,  but  also  that  depos- 
ited between  the  muscle  fibres  of  the  organ  itself.  In  the  case 
of  the  kidney  fat,  only  that  immediately  surrounding  the  organ 
was  taken. 

Apparently  in  this  tissue  and  under  the  influence  of  the 
rations  used  there  was  a decided  constancy  of  composition  main- 
tained. So  far  as  we  are  aware,  no  complete  data  are  available 
on  the  exact  proportion  and  kinds  of  neutral  fats  and  fatty 
acids  in  the  oils  of  the  seeds  and  roughage  used.  Hopkins 22 
gives  the  neutral  fat  content  in  corn  oil  as  3.66  per  cent  of 
stearin;  44.85  per  cent  of  olein,  and  48.19  per  cent  of  linolein. 
Stellwag23  gives  the  per  cent  of  free  fatty  acid  in  corn  grain 


21  Abderhalden  and  Samuely,  Zeit.  fur  Physiol.  Chem.  1905,  46,  p.  193. 

22  in.  Expt.  Sta.  Bui.,  53. 

23  Land,  versuch  Sta.,  37,  p.  134. 


Effect  of  Rations  Balanced  from  Restricted  Sources.  175 

as  6.7;  wheat  grain,  14.35;  oat  grain,  27.6.  Neutral  fat  in  corn 
grain,  88.7;  wheat  grain  78.73;  oat  grain,  61.6. 

The  difference  in  the  nature  of  the  oils  of  these  seeds  may 
not  be  great  enough  to  strongly  charactenze  the  one  from  the 
other  (on  this  point  we  have  no  data  as  yet),  nevertheless,  the 
nature  of  these  fats  is  so  markedly  different  from  those  stored 


Table  XV  Chemical  and  Physical  Constants  of  Carcass  Fats 


Corn. 

1 

Wheat. 

Oats. 

Mixture. 

Melting:  Point 

Omentum  fat 

47.5° 

44.7° 

46.4° 

45  7° 

Mesentery  fat .' 

50.0° 

50.8° 

50.9° 

50.1° 

51.0° 

Heart  fat 

50.1° 

48.0° 

Kidney  fat 

46.5° 

44.7° 

48.2° 

46.2° 

Koettstorfer  No. 

Omentum  fat 

191.3 

192.7 

190.4 

191  9 

Mesentery  fat 

190.8 

189.5 

192.3 

191.8 

190.7 

Heart  fat 

191.8 

191.7 

Kidney  fat 

191.4 

192.8 

189.4 

192.4 

Hiibl  No. 

Omentum  fat 

35.41 

36.7 

34.83 

36.03 

Mesentery  fat 

30.57 

27.11 

26.29 

27.30 

29.81 

Heart  fat 

37.20 

30.44 

Kidney  fat 

32.06 

33.05 

31.03 

29.32 

Reichert  No. 

Omentum  fat 

.75 

.75 

.70 

.89 

Mesentery  fat 

.48 

.32 

1.27 

.33 

.88 

Heart  fat 

.75 

.90 

Kidney  fat 

.63 

.63 

.62 

.63 

Refractive  Index  40° 

Omentum  fat 

Mesentery  fat 

1.4581 

1.4576 

1.4579 

1.4581 

1.4584 

1.4578 

1.4579 

1.4583 

1.4571 

1.4573 

Heart  fat 

1 . 4584 

Kidney  fat 

1.4573 

1.4585 

1.4576 

1.4578 

in  the  tissues  of  the  cow  that  very  important  synthetical  pro- 
cesses must  have  been  active  in  their  deposition.  The  fats  in  the 
tissue  of  the  animal  are  made  up  principally  of  stearin,  palmitin 
and  olein,  while  the  oils  of  the  feeding  stuffs  are  principally 
olein,  -and  the  higher  unsaturated  fats.  In  these  experiments, 
the  continued  feeding  of  com  did  not  make  a body  fat  with  a 
higher  content  of  unsaturated  fats  and  of  lower  melting  point 
than  the  continued  feeding  of  a mixed  ration,  nor  were  these 
constants  for  the  corn  animal  fats  unlike  those  given  by  Lew- 
kowitsch24  for  normal  beef  fat.  There  is  a good  deal  of  sub- 
stantial evidence  that  a large  intake  of  fat  of  a character  de- 
cidedly different  from  that  normal  to  the  body  will  modify  the 
constitution  of  the  fat  being  deposited,  and  the  results  of  Shutt 


24  Chemical  Technology  and  Analysis  of  Oils,  Fats  and  Waxes,  Vol. 
2,  pp.  813-14. 


176 


Wisconsin  Experiment  Station. 


indicate  that  there  is  an  influence  of  a constant  corn  diet  on  the 
character  of  the  fats  deposited  by  swine.  Nevertheless,  it  ap- 
pears that  the  fatty  tissue  of  the  cow  will  maintain  a uniform 
and  constant  composition  even  when  the  nature  of  the  fat  in  the 
feed  is  entirely  different  in  constitution  from  that  normally  de- 
posited. Only  when  the  fat  intake  is  excessive  may  the  nature 
of  the  fats  deposited  be  considerably  changed. 

Reaction  of  the  Urines 

General  differences  in  the  appearances  of  the  several  lots  of 
animals,  coupled  with  a decided  difference  in  the  character 
of  the  offspring  and  capacity  for  milk  secretion,  led  to  a fuller 
study  of  the  reaction  and  the  distribution  of  nitrogen  and  sul- 
phur in  the  urines  and  to  their  content  of  base  and  acid 
radicals.  It  was  believed  that  some  essential  differences  in 
this  respect  between  the  several  lots  might  suggest  causal  re- 
lations between  the  effect  of  the  rations  and  the  disturbed 
physiological  processes. 

In  the  metabolism  work  of  1909  the  reaction  of  the  urines 
was  determined  by  the  two  indicators  litmus  and  phenolphthal- 
ein.  The  urines  from  all  animals  on  all  rations  were  invari- 
ably acid  to  phenolphthalein.  The  wheat  ration  urines  alone 
were  acid  to  litmus..  The  urines  from  all  the  other  lots  were 
alkaline  to  litmus.  We  must  grant  that  the  color  changes  by 
indicators  depends  quite  as  much  on  the  chemistry  of  the  indi- 
cator as  on  the  constitution  of  the  medium  to  be.  tested  and  is 
significant  only  for  comparative  results.  It  is  in  this  way 
that  the  data  are  used  and  they  must  indicate  essential  dif- 
ferences in  the  urines.  A few  of  the  data,  but  representative  of 
all  obtained  at  other  periods  of  the  animal ’s  • life,  are  given  in 
Table  XVI. 

The  fact  that  stands  out  most  prominently  in  Table  XYI  is 
the  acid  character  to  both  indicators  of  the  urine  of  the  wheat- 
fed  group.  This  acidity  may  be  due  to  a deficiency  of  base  in  the 
ration,  with  the  consequent  formation  of  acid  salts  in  the  urine ; 
to  the  presence  of  organic  acids  in  the  urine  in  non-neutralized 
condition ; or  possibly  to  both  factors.  Reference  to  the  table  of 
intake  of  inorganic  constituents  by  the  wheat-fed  group  reveals 
the  fact  that  the  calcium,  magnesium,  and  potassium  content  of 
that  ration  w*as  lower  than  any  of  the  others ; the  silica  content 


Effect  of  Rations  Balanced  from  Restricted  Sources.  177 


was  higher;  and  the  other  acid  radicals  were  nearly  alike  in  quan- 
tity. Specific  determinations  of  the  inorganic  constituents  of  the 
urine  from  the  several  lots  showed  that  the  relation  of  bases 
to  acids  in  the  corn  ration  urines  was  approximately  as  3.5 
is  to  1:  in  the  oat  urine  as  2.5  is  to  1;  while  the  relation 
in  the  wheat  ration  was  as  1.5  is  to  1.  These  deductions  are 
based  on  actual  determinations,  from  which  the  bases  and  acids 
are  reduced  to  their  hydrochloric  acid  equivalents.  The  higher 
basicity  of  the  com  urine  also  favored  the  formation  of  car- 
bonates, which  were  present  in  those  urines  in  considerable 
quantity.  The  wheat  urines  were  free  from  carbon  dioxide. 

Table  XVI  Reaction  of  Urines 


Ration. 

No.  of 
animal. 

Date 

,1909. 

Yol. 

urine 

e,c. 

Reaction 

litmus. 

Reaction 

phenol- 

phthalein. 

Total  c,c.  N/ 10 
NaOH, 
Phenol. 

Jan. 

Corn 

558 

20 

3,041 

Alkali.... 

Acid 

58.5 

558 

21 

3,219 

Alkali.... 

Acid 

77.2 

rwn  * 

572 

20 

3,192 

Alkali 

Acid 

38.4 

Corn 

572 

21 

2,650 

Alkali.... 

Acid 

42.4 

Wheat 

585 

9 

3,6  2 

Acid 

Acid 

289.6 

Whftflt, 

565 

10 

2,848 

Acid 

Acid 

191.7 

Whpnt 

571 

9 

2.355 

Acid 

Acid 

164.0 

Wheat 

571 

10 

2,627 

Acid 

Acid 

261.0 

Oats 

567 

30 

3,054 

Alkali 

Acid 

54.9 

Oats 

567 

31 

4,820 

Alkali.... 

No  end  point. 

Oats  

569 

30 

3,477 

Alkali 

Acid 

52.2 

Oats 

569 

31 

5,503 

Alkali 

Acid 

55.0 

Mixture 

563 

Feb. 

10 

3, 113 

Alkali 

Acid 

124.0 

Mixture 

563 

11 

1,744 

Alkali.... 

Acid 

7.0 

Mixture 

555 

10 

7,587 

Alkali.... 

Acid 

27.7 

Mixture 

555 

11 

3,899 

Alkali.... 

Acid 

38.9 

The  excretion  of  Si  02,  which  was  contained  in  the  urine 
from  all  the  rations  in  uniform  amounts,  amounted  to  between 
2 and  3 grams  daily.  This  fact  removes  silica  from  the  possible 
factors  directly  influencing  the  reaction  of  the  urine. 

The  first  indication  of  positive  acidity  in  the  wheat  urines 
suggested  that  the  depressed  condition  of  this  group  wjais  the 
result  of  acidosis.  Generally  it  is  assumed  that  such  a con- 
dition will  lead  to  a larger  formation  of  ammonia  in  the  tis- 
sues for  the  purpose  of  maintaining  neutrality  or  slight  alka- 
linity and  a subsequently  increased  ammonia  excretion  in  the 
urine.  There  was,  however,  no  larger  absolute  quantity  of 
ammonia  nor  a larger  proportion  of  the  daily  nitrogen  excreted 
as  ammonia  in  the  urine  of  the  wheat-fed  animals,  as  compared 
with  the  com  or  other  groups.  Reference  to  Table  XYII  will 
show  the  evidence  for  this  conclusion. 


Wisconsin  Experiment  Station. 

The  theory  of  a possible  relation  of  low  base  intake  of  the 
wheat  ration  to  lowered  vitality  and  acid  urine,  was  subjected 
to  experimental  investigation  during  the  entire  gestation  period 
of  1909  and  1910.  To  two  of  the  animals  of  that  lot,  Nos. 
565  and  571,  there  was  ferd  daily,  in  addition  to  their  usual 
ration,  a salt  mixture  consisting  of  15  grams  calcium  carbonate 
(CaCO,),  47  grams  of  potassium  carbonate  (K2C03)  and  seven 
grams  magnesium  carbonate  (MgC03).  From  analyses  of  the 
corn  ration,  these  additions  to  the  wheat  ration  would  supply, 
with  an  intake  of  14  pounds,  an  exactly  comparable  quantity 
of  calcium,  magnesium,  and  potassium.  So  far  as  the  urines 
were  concerned,  their  reaction  to  litmus  was  gradually  changed 
to  an  alkaline  reaction. 

Unfortunately  No.  565  strangled  to  death  in  her  stall  on 
January  3,  1910,  consequently  giving  us  no  record  of  this  in- 
fluence of  higher  base  intake  on  calf  production. 

With  No.  571,  the  calf  born  was  a male  and  weighed  but 
57  pounds.  It  was  weak,  unable  to  get  up  and  suckle  its 
mother,  and  lived  but  22  hours.  So  far  as  our  observations 
with  these  cows  are  concerned,  the  added  alkaline  mixture  and 
mere  maintenance  of  an  alkaline  reaction  in  the  urines,  had 
no  influence  whatever  on  the  character  of  the  progeny  and  gen- 
eral restoration  of  vigor  to  the  mothers. 

We  must  recall,  in  this  connection,  the  experiment  of  Lunin  25 
with  mice  where  with  a food  mixture  consisting  of  purified 
proximate  constituents  and  the  ash  of  milk,  growth  could  not 
be  long  sustained.  This  failure  is  often  attributed  to  the 
fact  that  the  ash  materials  were  in  inorganic  combination. 

On  the  other  hand,  the  nearest  approach  to  an  answer  to  one 
phase  of  this  subject  of  the  adequacy  of  inorganic  salts  for 
constructive  functions,  is26  our  experiment  on  swine  where  a 
growing  sow,  receiving  a ration  too  low  in  total  phosphorus 
for  successful  growth,  was  able  to  grow  and  reproduce  with 
complete  maintenance  of  good  health  and  vigor  when  the  ad- 
ditional phosphorus  supply  was  drawn  wholly  from  inorganic 
phosphates.  The  young  produced  by  this  animal  also  appeared 
normal  in  every  respect.  In  addition,  the  experiments  of  McCol- 
lum 27  with  rats,  where  the  diet  again  consisted  of  simple  prox- 


25  Zeit.  f.  Physiol.  Chemie,  188i,  Vol.  5. 

26  Res.  Bui.  1,  Wis.  Expt.  Sta. 

27  Res.  Bui.  8,  Wis.  Expt.  Sta. 


Effect  of  Rations  Balanced  from  Restricted  Sources.  179 


imate  food  constituents,  (with  at  least  only  traces  of  ash  left  in 
them)  and  inorganic  salts,  but  provided  with  flavoring  material 
growth  was  sustained  for  a long  time.  Apparently,  if  proper 
intake  of  food  can  be  secured,  the  inorganic  bases  and  phos- 
phates, in  simple  salt-like  combinations,  can  perform  the  same 
junction  as  when  they  are  a part  oi  the  organic  structures  of 
the  ration.  More  data,  however,  are  required  in  respect  to  the 
bases ; and  the  conditions  of  combination  of  calcium,  magnesium 
potassium,  etc.,  for  optimum  effect  should  be  determined.  A 
priori,  one  can  believe  that  the  acid  of  the  gastric  juice  forms 
simple  salts  of  these  bases  existing  in  various  combinations  in 
the  feeds,  and  from  such  combinations  the  absorption  would  take 
place , but  no  data  on  this  point  are  yet  available.  This  labora- 
tory is  at  present  engaged  in  further  investigations  on  this 
subject. 


While  this  amount  of  evidence  should  not  warrant  final  con- 
clusions, nevertheless,  with  the  records  accumulated  since,  it 
appears  that  the  essential  differences  accruing  from  the  two 
rations  with  the  extreme  effects,  namely  com  and  wheat,  are 
not  to  be  attributed  directly  to  a lack  of  inorganic  base  material 
in  the  wheat  ration. 

There  was  a possibility  that  the  higher  silica  content  of  the 
wheat  ration  was  a factor  in  determining  the  path  of  excretion 
of  the  bases,  thus  tending  to  prevent  their  absorption  and 
availability  for  maintaining  a neutral  or  alkaline  reaction  in 
the  tissues  and  urine.  The  fact,  however,  that  nearly  all  of 
the  bases  of  wheat  straw  can  be  removed  from  their  combination 
by  .2  per  cent  hydrochloric  acid  (unpublished  data)  would 
hardly  warrant  such  an  assumption.  Further,  attempts  to  con- 
trol 'the  path  of  elimination  of  the  bases  by  administering  col- 
loidal silicic  acid  with  the  com  ration  failed  to  affect  the  re- 
action of  the  urine  of  these  animals.  Their  urine  always  re- 
mained -alkaline  to  litmus  even  where  the  administration  of 
silicic  acid  was  continued  for  two  or  three  months. 


These  experiments  on  ash  metabolism  and  reaction  of  the 


urines  are  as  yet  incomplete  and  all  deductions  advanced  for 
a possible  explanation  of  our  results  are  at  this  time  entirely 
tentative. 


It  was  suspected  that  the  depressed  condition  of  the  wheat-fed 
animals  and  somewhat  lessened  vigor  of  the  oat  and  mixture 
lots  might  be  due  to  the  formation  of  some  toxic  body  of  acid 


180  Wisconsin  Experiment  Station. 

nature,  formed  either  in  the  tract  or  during  metabolism  with 
its  possible  accumulation  in  the  tissues  and  final  elimination  in 
the  urine.  With  this  in  mind,  determinations  of  oxalic  acid 
were  made  on  the  day’s  urine  of  an  individual  from  each  lot 
over  a period  of  several  days  with  the  following  results.  The 
average  amount  of  oxalic  acid  in  one  day’s  urine  of  the  corn- 
fed  lot  was  .301  gram;  of  the  wheat-fed  lot,  .466  gram;  oat- 
fed  lot,  .285  gram;  while  those  fed  the  mixture  showed  .616 
gram. 

Tt  is  not  assumed  that  the  oxalic  acid  in  the  urine  was  free 
although  in  the  wheat  urine  it  is  possible  that  it  was  in  a free 
state.  The  general  presence  of  this  body  in  all  lots  precludes 
the  possibility  of  its  direct  relation  to  the  problem  of  lowered 
vigor  of  certain  groups  as  compared  with  others.  This  con- 
clusion in  regaid  to  oxalic  acid  by  no  means  exhausts  the  pos- 
sibilities of  the  presence  of  other  toxic  materials  formed  either 
in  the  tract  or  in  cellular  metabolism,  with  a failure  to  be  com- 
pletely eliminated.  Nor  is  it  necessary  to  assume  that  a pos- 
sible toxicity  resides  in  the  acid  character  of  such  substances. 
Many  materials  are  toxic  even  as  neutral  salts. 

In  this  direction  our  work  is  still  incomplete  and  fragmen- 
tary and  no  further  results  secured  in  this  direction  will  be 
reported  at  this  time. 

Nitrogen  Distribution  in  the  Urine 

In  the  first  year  of  the  experiment,  1907-08,  which  repre- 
sents a period  of  growth  only,  and  in  the  second  year  1908-09, 
representing  both  growth  and  reproduction,  the  distribution 
of  nitrogen  in  the  urine  of  the  different  lots  was  determined. 
The  data  presented  for  1907-08  represent  the  average  analyses 
of  two  successive  days  collection  from  one  individual  in  each  lot, 
and  are  expressed  as  per  cents  of  the  total  nitrogen  of  the 
urine.  The  methods  of  analyses  used  were  those  generally 
accepted  as  most  accurate  by  the  best-  authorities.  Uric 
acid  and  purins  were  estimated  by  the  Kruger-Schmidt  method ; 
urea,  creatin,  creatinin  and  ammonia  by  the  Folin  methods; 
hippuric  acid  by  the  method  of  Bunge  and  Schmiedberg28  and 
allantoin  by  the  method  of  PoduschRa  as  modified  by  Under- 
hill and  Kleiner.29 


28  Zeit.  Physiol.  Chem.  (1879)  Bd.  3. 

29  Jr.  Biol.  Chem.  (1908)  Vol.  4,  p.  166. 


Effect  of  Rations  Balanced  from  Restricted  Sources.  181 


The  results  secured  are  given  in  Table  XVII. 

In  1908-09  the  work  in  this  direction  was  again  continued 
with  analyses  of  the  daily  urine  collected  from  two  individuals  in 
each  lot  and  over  a period  of  seven  days.  The  observations 
were  made  again  on  the  same  animals  as  used  in  the  experiments 
of  1907  and  1908.  The  data  for  1908-09  represent  the  average 
for  the  entire  period  for  each  individual. 

Table  XVII  Nitrogen  Distribution  in  Urine 


Animal. 


Per 

cent. 


Per 

ppnt 


am- 

monia 


lit? 11  u 

urea 


Per 

cent. 

uric 

acid 


Per 

cent. 

purine 

base 


Per 
cent, 
hip- 
pur  ic 
acid 


Per 

cent. 

crea- 

tinin 


Per- 

cent. 

creatin 


Per 

cent 

allan- 

toin. 


N 


N 


N 


N 


N 


N 


N 


N 


1907-08. 

Corn 

...572 

2.11 

Wheat.... 

1.75 

Oat 

...567 

1.15 

Mixture .. 

...555 

2.68 

62.65 

80.05 

70.89 

71.21 


.46 

.39 

.37 

.60 


.035 

.061 

.033 

.024 


7.56 

1.57 
2.30 
3.46 


9.09 

3.01 

2.91 

4.58 


1.69 

2.81 

1.27 

1.37 


5.89 

0.00 

6.50 

9.21 


1908-09. 
Corn 

...572 

.31 

76.18 

Corn 

...558 

.40 

74.85 

Wheat . . . 

.39 

81.04 

Wheat . . . 

..571 

.34 

81.98 

Oat 

.24 

80.35 

Oat 

.26 

79.45 

Mixture . . 

.48 

74.07 

Mixture . . 

...563 

.34 

76.48 

.45 

.53 

.35 

.57 

.41 

.58 

.59 

.51 


.048 

6.26 

6.48 

.046 

5.94 

6.09 

.036 

5.36 

5.24 

.041 

1.91 

5.84 

.037 

3.54 

6.13 

.033 

3.83 

4.07 

.056 

4.19 

6.07 

.058 

4.15 

6.76 

3.71 

2.85 

2.91 

3.37 

3.98 

3.82 

6.01 

5.68 


3.47 

3.98 

2.89 

4.37 

6.21 

3.98 

3.68 

3.70 


The  most  striking  difference  in  the  forms  of  nitrogen  oc- 
curring in  these  urines  was  the  absence  of  allantoin  from  the 
urine  of  the  wheat-fed  animals  during  the  period  of  1907-08. 
There  are  quantitative  differences  among  the  other  constituents, 
for  example,  the  much  lower  liippuric  acid  and  creatinin  con- 
tent of  both  the  oat  and  wheat  urines  as  compared  with  the 
urines  from  the  corn-fed  animals— but  in  most  other  respects 
the  differences  in  nitrogen  distribution  are  not  large.  The  ab- 
sence af  allantoin  from  the  urine  of  this  one  individual  in  the 
wheat  lot,  after  its  first  observation,  was  repeatedly  confirmed 
not  only  for  that  individual,  but  for  all  others  in  the  group. 
At  times  the  quantity  separated  showed  .008  to  .010  gram 
nitrogen  as  allantoin  in  a volume  of  200  c.  c.  urine,  while  in  the 
other  lots  the  same  volume  gave  determinations  amounting  to 
.300  to  .500  gram  nitrogen.  It  is  entirely  probable  that  the 
method  for  allantoin  separation  is  not  refined  enough  to  prevent 
slight  precipitation  of  other  nitrogenous  substances  with  the 
reagents  used  in  the  allantoin  separation.  Our  conclusion  at 


182 


Wisconsin  Experiment  Station. 


present,  at  least,  is  that  this  body  was  absent  from  the  urine  of 
the  wheat-fed  calves  at  this  period  of  growth. 

The  origin  of  allantoin  among  the  products  of  cell  metabol- 
ism is  very  generally  ascribed  to  the  oxidation  of  uric  acid. 
Mendel  and  White  30  have  shown  that  after  intravenous  injection 
of  urates,  an  increased  elimination  of  allantoin  takes  place  in 
dogs  and  cats.  Apparently  this  particular  phase  of  metabolism 
was  suspended  or  interfered  with  in  this  group  of  animals.  The 
real  significance  of  its  absence  here  is  at  present  unknown,  but 
it  indicates  that  certain  processes  of  oxidation,  either  in  the  tis- 
sue cells  or  in  special  organs,  were  in  a degree  suspended. 

In  the  analyses  made  on  the  urines  in  the  year  1908-09  and 
which  represent  the  first  period  of  gestation,  allantoin  was 
found  in  the  urine  of  the  wheat-fed  calves,  as  well  as  in  all 
other  urines.  The  fact  that  the  animals  were  with  calf  during 
this  period  of  urine  collection  may  account  for  the  presence  of 
this  body  in  the  urine  of  the  wheat  group  at  this  time.  In  fact, 
allantoin  was  first  found  in  the  allantoic  fluid  of  the  cow  by 
Vanquellin  31  and  Lassagine  32  and  its  presence  in  the  urine  of 
the  mother  is  believed  to  have  had  its  origin  in  the  metabolism 
of  the  foetus. 

For  further  comparison  the  average  total  nitrogen  and  cre- 
atinin  nitrogen  output  in  grams  per  day  for  the  several  animals 
are  shown  in  Table  XVIII.  The  number  of  days  involved  is 
the  same  as  for  the  collection  of  the  data  of  the  preceding  table. 
Table  XVIII  Nitrogen  and  Creatinin  Output  in  Grams  Per  Day 


Feed. 

Animal  No. 

Total  nitrogen 
grams. 

[ Creatinin  ni" 
trogen  grams- 

Cnrn 

1907-1908. 

572 

27.38 

2.07 

Wheat 

565 

67.88 

2.04 

Oat 

567 

56.58 

2.08 

Mi  vhire 

555 

62.01 

2.83 

Corn 

1908-1909. 

572 

51.1 

3.08 

3.67 

558 

53.0 

Wheat 

565 

50.9 

2.76 

571 

46.2 

2.55 

Oat 

567 

46.2 

3.42 

569 

43.0 

4.79 

Mivturp  . 

555 

54.8 

4.76 

563 

37.7 

3.61 

so  American  Journal  of  Physiology,  No.  12,  p.  85. 
3i  Ann.  de  Ch.  und  Pharm.,  33-269. 

32Annal.  de  Chem.  et  Phys.,  17-301. 


Effect  of  Rations  Balanced  from  Restricted  Sources.  183 

During  the  period  of  greatest  growth,  1907-08,  the  creatinin 
output  in  the  urine  of  the  several  animals  was  much  alike.  On 
creatinin  free  diets  such  as  here  used,  it  must  be  assumed  that 
this  body  had  its  origin  wholly  in  endogenous  metabolism.  If 
the  creatinin  output  is  a fairly  accurate  measure  of  the  mass  of 
active  protein  tissue  of  an  animal,  as  appears  to  be  experiment- 
ally supported  by  other  investigations,  such  as  those  of  Folin  33 
and  one  of  the  authors, 34>  then  it  is  evident  that  these  animals 
at  this  stage  of  theit’  growth  must  have  contained  fairly  simi- 
lar quantities  of  protein  tissue.  Unfortunately  this  phase  of  the 
work  was  not  developed  at  this  stage  of  the  experiment  and  can- 
not be  supported  by  such  evidence  as  a slaughter  test. 

A year  later  when  the  urines  were  again  examined,  no  signi- 
ficant differences  in  respect  to  creatinin  output  appeared  among 
the  animals  of  the  several  groups.  At  this  time  the  creatinin 
index  of  the  protein  content  of  these  animals  again  indicates  a 
fairly  close  approximation  to  uniformity.  The  figures  on  the 
wheat  ration  are,  as  a group,  lower  than  all  others,  but  the  pres- 
ent state  of  our  knowledge  and  the  slight  variations  in  the  fig- 
ures would  hardly  warrant  the  conclusion  that  the  protein  con- 
tent of  these  animals  was  appreciably  lower  than  that  of  the 
other  groups. 

Sulphur  Metabolism 

Results  secured  on  the  nitrogen  distribution  in  the  urine  led 
us  to  a study  of  the  amounts  of  the  several  forms  of  sulphur 
normally  occurring  in  the  urine.  There  was  a possibility  that 
oxidation  processes  in  the  wheat-fed  mothers  were  less  vigorous 
than  in  the  corn-fed  group  and  that  such  a condition  would 
lead  to  different  quantitative  relations  between  the  forms  of 
sulphur  excreted  in  the  urine.  The  results  were  secured  on  the 
urines  collected  during  the  metabolism  period  of  1908 — 09  and 
are  expressed  in  per  cents  of  the  total  sulphur  of  the  urine  in 
Table  XIX.  The  methods  used  were  those  outlined  by  Folin. 
The  data  represent  averages  of  the  daily  analyses  of  the  urine 
collected  for  seven  days  from  two  individuals  in  each  lot.  The 
total  intake  of  sulphur,  as  well  as  the  average  daily  total  sulphur 
in  the  urine  is  given  in  grams. 


33  American  Jour.  Physiology,  1905,  Vol.  3. 

34  McCollum,  Wis.  Exp.  Sta.  Unpublished  data. 


Wisconsin  Experiment  Station. 


184 


Compared  with  35  human  urine  the  per  cent  of  neutral  or  un- 
oxidized sulphur  in  these  urines  is  very  large.  Thus,  Folin 
found  in  the  human  urines  studied  that  this  form  amounted  to 
3.7  to  6.3  per  cent  of  the  total  urinary  sulphur  and  that  the 
inorganic  sulphur  constituted  85  to  89  per  cent  of  the  total,  the 
rest  being  ethereal  sulphur. 


Table  XIX  Distribution  of  Sulphur  in  the  Urines  1908-09 


No,  of  Cow. 

Total  intake 
grams. 

Total  grams 
in  urine 

Per  cent 
inorganic. 

Per  cent 
ethereal. 

Per  cent 
neutral. 

Corn 

572 

13.27 

5.86 

22.3 

50.3 

27.4 

558 

13.27 

5.26 

29.6 

40.3 

30.1 

Wheat 

565 

12.30 

5.08 

32.5 

43.5 

23.9 

571 

12.30 

4.35 

21.1 

58.9 

20.1 

Oats 

567 

12.68 

6.35 

60.4 

23.6 

15.9 

569 

12.68 

6.52 

56.1 

25.3 

18.6 

Mixture 

555 

12.75 

6.44 

43.0 

30.2 

16.8 

563 

12.75 

4.86 

50.0 

26.9 

23.1 

From  these  results  there  is  nothing  to  indicate  a lower  de- 
gree of  oxidation  of  the  sulphur  in  the  metabolism  of  the  wheat- 
fed  animals  than  in  the  corn-fed  animals.  In  all  other  urines 
the  per  cent  of  neutral  sulphur  was  not  greatly  different  and 
distinct  enough  to  characterize  them  from  the  corn  or  wheat 
urines.  Ethereal  sulphur  is  high  in  amount  as  compared  with 
human  urine  and  indicates  a larger  production  and  absorption 
from  the  tract  of  this  class  of  animals  of  aromatic  compounds 
formed  through  putrefaction.  The  production  of  this  class  of 
compounds  appears  to  have  been  higher  in  the  corn  and  wheat 
urines  than  in  the  urines  of  the  oat-  and  mixture-fed  groups,  but 
between  the  corn  and  wheat  urines  there  was  practically  no  dif- 
ference. It  should  be  remembered  that  the  rations,  including 
roughage  as  here  used,  may  contain  a considerable  portion  of 
the  sulphur  intake  as  sulphates,  while  it  is  probable  that  these 
forms  are  very  small,  if  present  at  all  in  the  grains.  This 
would  modify  in  a degree  any  conclusion  that  inorganic  and 
ethereal  sulphur  orginate  wholly  from  unoxidized  sulphur  in 
rations  of  the  character  here  used. 


35  Folin,  Amer.  Jour.  Physiol.  1905,  13. 


Effect  of  Rations  Balanced  from  Restricted  Sources.  185 


Effect  of  a Change  of  Rations 

On  May  26,  1910,  after  a record  of  three  years,  eleven  of  the 
herd  remained.  No.  566  of  the  corn  lot  was  at  this  timo 
slaughtered  for  carcass  samples.  On  the  above  date,  the  ten  re- 
maining animals  were  shifted  to  new  rations.  It  is  a significant 
fact  that  we  had  not  lost  a single  animal  from  the  corn-fed 
group  from  disease  or  from  infection,  or  other  difficulties  arising 
at  calf  birth.  All  other  lots  had  suffered  some  depletion  from 
one  cause  or  another.  One  case  of  anthrax  occurred  in  the  wheat 
lot  and  another  animal  in  the  same  lot  was  found  dead  in  her 
stall,  death  probably  having  been  due  to  strangulation. 

One  of  the  mixture  lot  developed  uterine  infection  after  calf 
birth  in  1910  and  was  ordered  killed  by  the  station  veterinarian. 
No.  568  in  the  oat  lot  suffered  from  the  same  trouble  and  was 
killed,  while  No.  569  of  the  same  group,  after  suffering  a good 
deal  of  difficulty  in  giving  birth  to  her  calf  was  killed  for  tissue 
samples. 

It  was  certainly  apparent  that  the  corn-fed  mothers,  in  their 
higher  states  of  vigor  and  nutrition,  were  able  to  carry  out  the 
processes  of  calf  birth  with  greater  normality  than  the  other 
groups  and  seemed  endowed  with  greater  powers  of  resistance. 

In  the  change  of  rations,  the  mixture  group  was  discontinued. 
It  had  not  served  its  supposed  function  of  providing  the  highest 
standard  and  consequently  was  of  no  further  use.  The  changes 
made  were  as  indicated  in  Table  XX. 


Table  XX  Changes  in  Rations 


To  Corn. 

To  Wheat. 

To  Oats. 

562  Formerly  Mixture 

555  Formerly  Mixture 

558  Formerly  Corn 

567  Formerly  Oats 

556  Formerly  Corn 

563  Formerly  Mixture 

570  Formerly  Wheat 

557  Formerly  Oats 
572  Formerly  Corn 

571  Formerly  Wheat 

The  rations  were  exactly  the  same  as  those  used  in  1909-10, 
providing  equal  amounts  of  digestible  protein  and  production 
therms  according  to  the  standards  of  Kellner  and  Armsby.  Re- 
ference to  Figures  20  to  22  will  show  the  condition  of  the  ani- 
mals at  the  beginning  of  the  change. 

The  extremely  emaciated  condition  of  No.  570  of  the  wheat 
lot  at  the  time  of  initiating  the  change  should  be  observed.  The 


186 


Wisconsin  Experiment  Station. 


. ^ % 


§’M# 


670  Wheat  567  Oats  562  Mixture 

Figure  20  Animals  changed  to  corn  May  31,  1910.  Upper  row  at  beginning  of  change:  second  row  nftcr 


— — — - ~ — — 

— . — — _ 


Effect  of  Rations  Balanced  from  Restricted  Sources.  18? 


<© . 
m cs 

vo 

w, 

vO  T3 


8 1-1 

CO 

s 


VO 


o 

Qj 

a! 

o —> 

.» 

^ M 
(5.^3 

o a 
o 

^g 

<u 

P-  K 
£■« 


188  Wisconsin  Experiment  Station. 


Figure  22  Animals  changed  to  oats  May  31,  1910.  Upper  row  from  left  to  right.  Nos  571,  603,  and  558,  at  beginning  of  change; 


Effect  of  Rations  Balanced  from  Restricted  Sources.  189 


change  to  the  new  ration  was  made  suddenly.  We  had  no  dif- 
ficulty whatever  in  obtaining  complete  consumption  of  the  new 
rations  by  all  individuals  except  those  changed  to  the  wheat 
ration,  and  here  the  difficulty  applied  more  particularly  at  first 
to  these  brought  over  from  the  corn  ration.  These  animals  re- 
fused to  consume  a proper  allowance  of  feed,  but  by  gradual 
substitution  of  small  amounts  of  the  wheat  ration  for  an  equiv- 
alent amount  of  the  withdrawn  corn  ration,  complete  change  was 
effected  by  June  10,  1910. 

One  of  these  animals,  No.  572,  received  in  addition  to  her 
ration,  the  mixture  of  alkaline  salts  already  mentioned  on  page 
178,  for  the  express  purpose  of  determining  again  the  effect  of 
maintaining  an  alkaline  urine  on  the  wheat  ration. 

Animals  Changed  to  Corn  Ration  Those  animals  changed  to 
the  corn  ration  maintained  their  appetites,  showed  no  discomfit- 
ure from  the  consumption  of  an  entirely  new  feed,  and  digested 
the  new  ration  at  least  as  completely  as  they  had  their  former 
ration.  This  can  be  seen  by  reference  to  Table  XXII.  These 
animals  manifested  no  symptoms  of  stiffness,  swollen  joints,  ina- 
bility to  walk,  or  general  muscular  rheumatism,  as  shown  in  vary- 
ing degrees  of  severity  by  all  the  individuals  in  the  other  two  lots 
They  all  went  through  their  year  in  improved  condition,  main- 
tained or  increased  their  weight,  reproduced  themselves  and  are 
at  this  writing  (summer  of  1911)  in  apparently  as  good  and  cer- 
tainly in  the  case  of  570,  a former  wheat-fed  animal,  in  better 
health  and  vigor  than  ever  before. 

Animals  Changed  to  Oat  Ration  The  animals  changed  to 
the  oat  ration  at  first  stood  the  change  well  and  made  good  pro- 
gress. In  July  1910,  No.  558,  formerly  corn,  sustained  an  in- 
jury  in  the  out-door  paddock  from  which  she  never  fully  re- 
covered and  this  necessitated  her  being  killed  in  October,  1910. 
However,  during  the  first  three  months  of  change  her  daily 
consumption  of  food  w'as  approximately  17  pounds,  but  gradu- 
ally declined  under  the  aggravation  of  her  injury. 

No.  571,  formerly  wheat,  suffered  from  the  change  of  ration 
with  swollen  joints  and  bloating.  For  six  months  her  average 
daily  consumption  of  feed  was  between  16  and  17  pounds,  but 
after  that  her  consumption  receded  until  her  bloated  and  stiffen- 
ed condition  became  so  acute  that  entire  loss  of  appetite  re- 
sulted. Previous  to  her  death  the  animal  was  unable  to  sup- 
port her  own  weight.  On  March  17  she  gave  birth  to  a calf 


190 


Wisconsin  Experiment  Station. 


weighing  but  32  pounds  and  born  58  days  ahead  of  the  cab 
culated  time.  On  March  20  she  died.  Post-mortem  examina- 
tion by  Dr.  F.  B.  Hadley,  station  veterinarian,  showed  that  the 
animal  had  septic  metritis  complicated  by  general  constitutional 
weakness. 

No.  563  remained  throughout  the  entire  period  from  June 
1910  to  June  1911,  in  fair  bodily  vigor  and  appetite.  She  ap- 
proximately maintained  her  body  weight,  the  record  showing  a 
loss  of  only  19  pounds  for  the  entire  period.  Only  at  one  period 
of  a few  weeks  duration  in  February  1911  and  just  before  par- 
turition did  she  manifest  any  symptoms  of  impaired  vigor.  At 
that  time  the  limbs  became  swollen  and  the  animal  showed  some 
difficulties  in  walking.  From  this  she  recovered  with  the  same 
ration  and  at  the  present  writing  is  in  good  health. 

Animals  Changed  to  Wheat  Ration.  The  animals  changed  from 
the  other  rations  to  this  ration  were  maintained  on  it  with  diffi- 
culty. Stiffness  and  swollen  joints,  inability  to  rise  from  a recum- 
bent position  and  eventually  a loss  of  appetite  were  mlet  with  con- 
stantly. Only  after  we  had  learned  that  the  progress  of  these 
symptoms  could  be  effectually  arrested  by  a return  to  the  corn 
ration  were  we  able  to  carry  two  animals  from  this  group 
through  the  entire  year  and  keep  them  on  the  wheat  ration  for 
a part  of  the  time. 

No.  556,  formerly  corn,  but  receiving  no  alkaline  salts,  showed 
such  symptoms  as  above  described,  on  September  7,  or  approxi- 
mately three  months  after  the  change  of  ration.  (See  Figure  23.) 
Up  to  that  time  she  had  consumed  daily  12%  pounds  of  the  ra- 
tion and  appeared  in  good  health.  Stiffness  in  the  joints  soon 
came  on  and  she  refused  to  move  from  her  recumbent  position ; 
on  September  14  the  alkaline  salts  were  administered.  No  positive 
effect  resulted  and  September  18  stimulants  were  given  by  Dr. 
Hadley.  This  effected  a partial  recovery  and  on  September  20 
the  animal,  with  help,  was  able  to  rise.  There  was  no  recovery 
of  appetite  and  on  September  30  she  grew  worse  and  on  October 
4 was  killed. 

Post-mortem  revealed  very  few  lesions  of  importance  except 
those  of  the  joints.  “The  organs  of  the  thoracic  cavity  were 
apparently  normal,  the  stomach  and  intestines  showed  no  in- 
dications of  disease,  neither  did  any  of  the  other  abdominal  vis- 
cera. In  several  of  the  joints  of  the  limbs  the  articular  cartilages 
showed  varying  degrees  of  involvment,  ranging  from  a dullness 


Effect  of  Rations  Balanced  from  Restricted  Sources.  191 


of  the  usually  shiny  articular  surface,  to  a rough,  velvety  and 
frequently  eroded  surface.  These  changes  were  confined  to 
areas  from  the  size  of  a pea  to  that  of  a dime  or  larger.  The 
bone  beneath  the  cartilages  appeared  more  or  less  inflamed.  At- 
tached to  the  synovial  membrane  lining  the  joint  capsule  of  the 
right  shoulder  joint  was  a pedunculated,  highly  congested  piece 
of  membrane  which  projected  into  the  joint  cavity.  These  find- 
ings account  for  the  inclination  of  the  animal  to  keep  off  its  feet 


as  much  as  possible.  It  can  be  safely  assumed  that  there  was 
no  infectious  or  contagious  disease  present. ” (Dr.  Hadley’s  Re- 
port.) 

No.  557  began  to  show  the  same  symptoms  of  stiffness  and  loss 
of  appetite  on  September  9,  1911.  Her  condition  was  even  more 
aggravated  than  that  of  No.  556  and  under  the  advice  of  the 
veterinarian,  stimulants  were  administered,  but  only  after  a 
lapse  of  several  days  from  the  first  observation  of  her  depressed 
condition.  This  condition  of  stiffness,  etc.,  did  not  in  the  case 
of  any  animal  come  on  suddenly,  but  was  apparently  a slowly 
accumulating  effect.  For  that  reason  changes  of  diet  in  these 
early  cases  were  not  resorted  to  early  enough  to  save  the  animal. 
It  was  soon  found  that  restoration  of  an  animal  to  the  corn  ra- 
tion would  be  followed  by  complete  recovery.  In  her  debilitated 


192 


Wisconsin  Experiment  Station. 


condition,  other  disturbances  followed  and  September  30,  she 
died  of  traumatic  pneumonia,  probably  contracted  from  the 
aspiration  of  some  foreign  material. 

No.  555  began  to  show  symptoms  of  swollen  .joints  and  stiff- 
ness September  10.  On  September  30  her  condition  was  some- 
what worse,  and  October  2 she  was  restored  to  the  corn  ration. 
On  October  19  the  swelling  and  stiffness  had  entirely  dis- 
appeared, and  cn  November  14  she  was  again  placed  cn  the 


Figure  24  No.  555.  Mixture.  Condition  five  months  after  chcnging  to  wheat. 


wheat  ration.  Her  appetite  wras  good  and  her  consumption 
averaged  12  to  13  pounds  of  feed  daily.  On  January  19  a re- 
occurrence of  symptoms  of  stiffness  and  swollen  joints  appeared. 
(See  Figure  24.)  On  January  27  she  was  again  placed 
on  the  corn  ration,  with  slow  recovery,  and  February  15  she  was 
again  in  apparently  normal  condition.  Our  inability  to  change 
a mature  animal  to  the  wheat  ration  and  have  her  live,  compelled 
us  to  continue  this  animal  on  the  corn  ration  for  the  rest  of  the 
experimental  period  of  1911.  Unfortunately  she  was  not  with 
calf. 

No.  572,  wheat  ration  plus  alkaline  salts,  also  began  to  show 
the  same  symptoms  on  September  17.  On  October  20  she  was 
placed  on  the  corn  ration,  with  entire  recovery.  After 
feeding  the  corn  ration  for  forty-five  days,  she  was  again  re- 
turned to  the  wheat  ration  and  alkaline  salts.  This  was  on 
December  3,  The  animal  manifested  no  further  return  of  unto- 


Effect  of  Rations  Balanced  from  Restricted  Sources.  193 

ward  symptoms  until  May  15,  1911,  when  she  was  again  taken  to 
the  corn  ration.  With  the  exception  of  the  period  of  forty-five 
days  on  the  corn  ration  she  was  carried  through  the  entire  period 
from  May  30,  1910,  to  May  15,  1911,  with  an  average  daily  con- 
sumption of  13.8  pounds  of  the  wheat  feed  mixture  plus  the 
alkaline  salts.  At  the  end  of  that  period  she  was  in  fairly  good 
condition  and  had  lost  during  the  year  but  59  pounds,  her  final 
weight  being  1023  pounds. 

Apparently  the  maintenance  of  an  alkaline  urine  had  been  of 
some  remedial  value  to  this  individual,  although  it  had  not 
entirely  prevented  the  occurrence  of  stiffness,  swollen  joints  and 
rheumatic  tendencies,  nor  maintained  a high  state  of  vigor  for 
reproduction  and  mammary  processes  as  shown  in  the  records 
of  calf  birth  and  milk  secretion. 

It  should  be  stated  that  mature  animals  from  other  rations, 
when  changed  to  the  wheat  ration,  developed  urines  acid  to 
litmus  after  a lapse  of  from  ten  days  to  two  weeks.  Those 
changed  from  wheat  to  corn  or  oats  produced  urines  neutral  or 
alkaline  to  litmus.  It  is  a remarkable  fact  that  we  could  not 
successfully  change  a mature  corn-  or  oat-fed  animal  to  the  wheat 
ration  and  have  her  maintain  health.  Only  when  alkaline  salts 
were  used  was  it  possible  to  carry  a single  individual  through 
the  year  on  the  wheat  ration,  and  even  that  individual  showed 
at  times  marked  effects  of  the  ration. 

On  the  other  hand,  it  was  possible  to  change  mature  animals 
accustomed  to  the  same  food  material  for  three  years  to  the  corn 
and  oat  rations.  Marked  improvement  in  well  being,  especially 
when  changed  to  the  corn  ration,  always  followed.  A corn-fed 
animal  passing  to  oats  showed  no  noticeable  symptoms  of  a set- 
back, but  we  have  no  records  of  the  effect  of  such  a change  on 
reproduction. 

In  Table  XXI  are  recorded  the  data  on  the  effect  of  the 
change  in  ration  on  the  live  weight  of  the  animals.  These  data 
cover  the  period  from  May  1910  to  May  1911. 

The  effect  of  the  change  of  ration  on  the  live  weights  of  the 
animals  was  not  uniform  in  direction  among  all  the  individuals 
excepting  with  those  on  the  wheat  ration.  In  this  group  we 
found  uniform  losses.  The  amount  of  loss  for  the  entire  period 
and  especially  for  three  of  the  animals  would  not  be  considered 
significant  or  indicative  of  starvation.  The  loss  of  189  pounds 
by  No.  557  is,  on  the  other  hand,  considerably  larger  than  for 


194 


Wisconsin  Experiment  Station. 


the  others  of  this  group,  and  accompanied  her  debilitated  con- 
dition manifested  by  stiffness  and  swollen  joints.  At  the  pre- 
sent writing,  No.  555  and  572  are  living  and  in  good  health  and 
apparent  vigor.  In  the  other  lots'  the  animals  either  approxi- 
mately held  their  own  weights,  or  as  in  the  ea:e  of  571,  actually 
made  a substantial  increase. 


Table  XXI  Gain  and  Loss  Due  to  Changed  Rations 


No.  cow 

Average 
dally  feed 
eaten  lbs. 

Final  weight. 

\ Gain  or  loss. 

Corn 

562 

14.3 

1,033 

+68 

567 

15  7 

1,064 

—29 

570 

15.3 

893 

+ 3 

Wheat 

13.8 

1,222 

—67 

556 

11.6 

1,095 

-41(a) 

557 

11.4 

960 

—189(b) 

572 

13.8 

1,023 

—59 

Oats 

558 

15.2 

1,161 

+37  (c) 

568 

13.4 

873 

—19 

571 

11.9 

1,312 

+261(d) 

(a)  Record  for  129  days,  (b)  Record  for  125  days,  (c)  Record  for  42  days,  (d) 
Record  for  294  days. 


It  is  always  of  interest  to  the  physiological  chemist  to  learn 
more  concerning  the  adaptability  of  the  digestive  juices  to  the 
kinds  of  feed  consumed.  It  was  questioned  whether  the  long- 
continued  sameness  of  diet  to  which  these  animals  were  sub- 
jected would  have  so  changed  the  character  of  the  digestive 
juices  as  to  seriously  influence  the  coefficents  of  digestibility  of 
the  new  feeds.  For  this  reason  such  coefficients  of  nitrogen  and 
dry  matter  were  again  determined  on  certain  individuals  dur- 
ing the  month  of  June  1910,  or  after  *a  lapse  of  two  weeks  from 
the  time  of  the  change  in  the  ration.  The  data  are  recorded  in 
Table  XXII,  and  represent  the  average  of  a three-day  record. 

Table  XXII  Coefficients  of  Digestibility  of  Nitrogen  and  Dry 

Matter 


1911 

1910 

N 

coefficient 

Dry  matter 
coefficient 

N 

coefficient 

Dry  matter 
coefficient 

Corn  570  formerly  wheat 

67.06 

66.70 

75.5 

68.83 

Corn  567  formerly  oats 

64.25 

60.04 

70.3 

69.02 

Wheat  556  formerly  corn 

75.70 

61.66 

Wheat  557  formerly  oats 

75.47 

61.05 

Effect  of  Rations  Balanced  from  Restricted  Sources.  195 

Where  available  for  the  same  individual,  data  on  the  old  ra- 
tions are  given.  , 

While  this  method  of  research  is  not  sufficiently  refined  to 
detect  slight  differences  in  the  character  of  the  digestive  juices, 
yet  in  the  main  they  do  indicate  that  the  rates  of  solution  of 
the  new  feeds  were  not  materially  different  from  those  of  the 
old  rations.  Of  course,  the  figures  in  the  table  do  indicate  some 
differences,  but  we  do  not  believe  that  they  are  of  such  a degree 
as  to  be  taken  seriously. 

The  records  of  reproduction  and  milk  secretion,  following 
the  change  of  the  rations  made  in  May  1910,  are  given  in  Table 
XXIII.  In  addition,  for  purposes  of  comparison,  the  data  for 
the  daily  milk  yield  for  1910  and  the  weights  of  the  calves  for 
1909  and  1910  are  also  added. 


Table  XXIII  Effect  of  Change  of  Ration  on  Calf  Production  and 

Milk  Secretion 


No.  of 
cow. 

Ration 

1911. 

Former 

ration. 

Wt. 

calf 

1911 

lbs. 

Calved 

before 

time, 

days. 

Wt. 

calves 

1909. 

wt, 

calves 

1910. 

Av. 

daily 

milk 

1911. 

Av. 

daily 

milk 

1910. 

562 

567 

570 

Corn 

Corn 

Corn 

Mix. 

Oat 

Wheat 

1 

65 

95 

81 

13 

0 

9 

55 

77 

48 

67 

70 

47 

24.6 

26.4 

19.2 

21.00 

27.90 

16.7 

555 

556 

557  1 

572  | 

Wheat 

Wheat 

Wheat 

Wheat 

! Mix. 
Corn 
Oat 
Corn 

(a) 

(b) 
(b) 
36 

18 

1 

85 

93 

12.9 

26.70' 

558  ] 

563 

57,  ; 

Oats 

Oats 

Oats 

Corn 

Mix. 

Wheat 

(b)  ! 

47 

32  ! 

2i  .3  ! 

17 

58 

65 

44 

66 

57 

(c) 

21.6- 

(a)  Not  with  calf,  (b)  Killed,  (.c)  Died  after  birth  of  calf. 


The  records  clearly  show  that  the  rations  had  an  influence  on 
the  character  of  the  offspring  and  milk  secretion,  even  in  a 
single  gestation  period.  This  important  and  remarkable  fact 
is  borne  out  particularly  in  the  changes  from  corn  to  wheat  and 
wheat  to  corn.  No.  570,  a former  wheat-fed  animal,  but  now 
receiving  corn,  carried  her  calf  nearly  to  the  calculated  time 
and  produced  an  animal  weighing  81  pounds,  as  compared  with 
one  of  47  pounds  and  another  of  48  pounds  in  the  two  previous 
years.  Unfortunately  this  calf  was  a malpresentation  and  suf- 
focated during  birth,  consequently  giving  us  no  judgment  of 
its  vigor  and  strength. 


196 


Wisconsin  Experiment  Station. 


The  calves  of  Nos.  562  and  567  were  both  strong  and  vigor- 
ous, suckling  the  mother  within  an  hour  after  birth  and  de- 
cidedly in  contrast  to  the  records  of  the  offspring  from  these 
mothers  in  1909  and  1910. 

The  volume  of  milk  secreted  by  these  corn-fed  animals  also 
showed  some  improvement  over  the  record  of  the  mixture  and 
wheat  rations  of  the  previous  year.  From  the  oat  ration  there 
Was  a slight  decline. 

The  calf  produced  by  the  wheat-fed  animal  No.  572,  changed 
from  corn,  and  receiving  alkaline  salts,  was  extremely  small 
and  born  dead.  It  was  carried  to  within  eighteen  days  of  the 
calculated  time  for  parturition  and  weighed  but  36  pounds,  as 
compared  with  the  former  record  of  93  and  85  pound  calves  on 
the  corn  ration.  Milk  secretion  was  reduced  one-half.  It  is 
again  necessary  to  emphasize  the  fact  that  her  daily  average 
consumption  of  food  for  the  year  was  13.8  pounds,  not  greatly 
unlike  that  consumed  by  the  corn-fed  mothers,  namely  about 
15  pounds. 

The  only  calf  produced  on  the  oat  ration  (that  of  No.  563), 
was  weak,  unable  to  stand,  and  required  nursing  from  the  bot- 
tle. It  lived  but  forty-eight  hours. 

This  record  of  the  change  of  rations  emphasizes  more  than 
anything  else  the  comparative  physiological  value  of  the  feeds. 
Certain  combinations  of  accepted  normal  feeds  were  decidedly 
effective  in  maintaining  vigor  and  health,  while  other  combina- 
tions of  feed  had  an  opposite  effect,  measured  in  ill  health,  poor 
condition,  under-sized  progeny,  and  lowered  activity  in  milk 
secretion.  That  such  effects  could  be  produced  in  a single  gest- 
ation period  whereby  strong  vigorous  progeny  were  changed 
to  undersized,  weak  offspring  by  a change  of  ration  alone  is  a 
remarkable  fact. 

It  is  also  apparently ^ evident  that  normal  rations  will  vary 
in  their  effect  on  the  powers  of  resistance  of  an  individual. 
Animals  in  poor  states  of  nutrition,  as  for  example,  the  wheat 
group,  showed  difficulties  in  giving  birth  to  their  young,  evid- 
ent by  retention  of  the  afterbirth,  or  a great  delay  in  its  ex- 
pulsion. 

Evidently  a “habit  of  metabolism’’  was  not  so  firmly  estab- 
lished by  the  long-continued  use  of  certain  feed  materials  as 
to  make  it  impossible  to  change  suddenly  the  nature  of  the 
animal’s  ration.  What  good  or  bad  effects  a ration  possessed 


Effect  of  Rations  Balanced  from  Restricted  Sources.  197 

were  in  time  shown  by  the  animals  in  spite  of  residuary  effects 
the  original  ration  had  left,  and  this,  as  above  stated,  was  appar- 
ent within  the  time  of  a single  gestation  period. 

Bearing  of  this  Work  on  Nutrition  Problems 

The  history  of  these  animals,  receiving  such  restricted  ra- 
tions, emphasizes  to  us  the  inadequacy  of  present  methods  of 
research  as  being  able  to  give  final  and  complete  answers  to  the 
question  of  the  comparative  nutritive  value  of  feeds.  Measur- 
ing the  total  digestible  nutrients  is  manifestly  incorrect,  be- 
cause it  takes  no  cognizance,  of  the  work  done  in  preparing  the 
material  for  absorption.  Further,  it  is  evident  from  our  ex- 
periments that  measuring  the  production  value  of  feeds  and 
the  formulation  of  rations  on  the  basis  of  their  digestible  pro- 
tein and  available  energy  is  also  inadequate.  Very  probably 
the  measurement  of  production  energy,  coupled  with  studies 
of  the  physiological  reactions  of  feeding  materials,  will  result  in 
definite  and  final  knowledge  in  regard  to  their  worth ; but  until 
we  have  records  of  the  influence  of  the  material  on  the  physiol- 
ogical status  of  the  animal,  such  standards,  based  on  digestible 
protein  and  available  energy,  must  be  accepted  only  tentatively. 

There  is  nothing  said  in  the  present-day  tables  of  commonly 
accepted  standards  to  prevent  one  from  making  up  a ration  for 
his  cow  from  wheat  straw,  wheat  meal,  and  wheat  gluten.  The 
experienced  feeder  may  feel  that  such  a ration  would  not  do,  but 
the  student  of  animal  nutrition  knows  that  he  can  at  least 
satisfy  the  mathematical  requirements  of  the  standard  by  the 
use  of  such  materials ; and  yet  we  here  have  definite  informa- 
tion that  such  a combination  of  materials  will  not  completely 
answer  the  physiological  requirements  of  the  breeding  cow.  It 
might  be  said  that  any  good  feeder  knows  that  there  should  be 
variety  in  the  ration  and  not  continued  sameness,  but  reference 
should  be  made  to  the  fact  that  the  corn  ration,  made  up  of 
corn  stover,  corn  meal  and  gluten  feed,  was  not  less  defective 
in  this  respect  than  the  wheat  ration. 

It  might  be  said  that  the  wheat  ration  really  had  more  same- 
ness and  monotony  than  the  corn  ration,  but  if  monotony  was  the 
source  of  the  trouble,  then  the  mixed  ration  should  have  been  our 
standard  instead  of  the  corn  ration. 

Nor  was  environment  the  factor  in  this  experiment,  because 
all  of  the  animals  were  kept  under  identical  conditions. 


19S 


Wisconsin  Experiment  Station. 


It  might  be  objected  that  the  mere  long-continued  feeding  of 
the  same  ration  to  this  class  of  animals  would  ultimately  bring 
about  depression  and  loss  of  vigor,  but  such  conditions  did  not 
obtain  with  the  corn  ration,  and  where  they  did  occur,  as  for 
example  with  the  wheat  ration,  the  results  must  be  attributed  to 
causes  other  than  mere  monotony  of  diet. 

If  consumption  of  material  had  been  interfered  with,  then 
monotony  of  diet  might  have  become  a factor  in  our  results. 
That  this  condition  did  not  obtain  is  evidenced  by  reference  to 
the  records  in  Table  III. 

It  is  true  that  our  rations,  fortunately,  so  exaggerated  their 
ultimate  effects  as  to  make  them  unmistakable.  In  this  respect 
the  use  of  materials  from  a variety  of  sources  may  apparently 
succeed  in  obscuring  more  or  less  the  good  or  bad  effects  of 
each  particular  source  of  the  materials  involved.  For  example, 
our  mixture  ration  gave  results  intermediate  in  effect  to  either 
the  corn  or  wheat  ration.  Obviously  the  good  effects  of  the  corn 
succeed  in  obscuring  more  or  less  the  good  or  bad  effects  of 
the  wheat  portion,  with  results  of  less  pronounced  character  in 
either  direction  as  compared  with  the  results  secured  when  these 
materials  were  fed  alone. 

It  appears  from  our  data  that  the  method  of  experimenting 
as  here  outlined  can  be  expected  to  give  results  of  definite  value. 
It  should  again  be  stated  that  this  preliminary  investigation 
suggests  a method,  with  some  preliminary  data  on  the  effect  of 
certain  rations,  rather  than  establishes  final  values  for  any  part 
of  these  rations.  Experimental  rations  can  be  so  constructed 
as  to  provide  proper  quantities  of  inorganic  material,  digestible 
protein  and  production  therms,  and  of  a character  (roughage  or 
not)  suited  to  the  species  of  animal  to  be  experimented  with. 
Parts  of  a plant  either  grain  or  straw,  can  make  up  the  whole 
ration,  or  only  part  of  it.  The  number  of  animals  used  must 
be  large  enough  to  obscure  all  initial  differences  in  individuals. 
The  period  of  observation  must  be  longer  than  commonly  sup- 
posed, if  we  are  to  establish  the  physiological  value  of  a feed 
material.  If  it  is  merely  for  growth,  then  the  period  may  be 
shorter, — possibly  six  months  to  a year, — but  such  a record  will 
not  establish  its  physiological  value  for  other  functions.  The 
necessity  of  knowing  the  effects  on  reproduction  and  milk  se- 
cretion are  so  imperative  that  the  animals  should  be  carried 
through  at  least  two  gestation  periods  on  the  same  ration  that 


Effect  of  Rations  Balanced  from  Restricted  Sources.  199 

-was  used  in  the  period  of  growth.  A ration  once  established  as 
of  high  excellence  in  effect  can  be  modified  by  the  substitution, 
for  a part  of  the  material,  of  the  substance  to  be  tested.  Variety 
of  combinations  can  thus  be  made.  The  materials  should  be 
fed  over  long  enough  periods  of  time  and  until  reliable  data 
are  secured.  This,  we  believe,  is  the  only  possible  way  by 
which,  in  the  present  state  of  our  knowledge  their  physiological 
values  can  be  established.  We  believe  that  the  establishment 
•of  these  physiological  values  for  a ration  must  supplement  the 
determination  of  their  production  values,  if  the  latter  are  to  be 
made  of  permanent  and  accurate  worth. 

Nor  can  it  with  perfect  safety  be  concluded  that  results  sc- 
oured with  one  species  of  farm  animal  can  be  directly  translated 
into  rules  for  feeding  all  others.  We  would  not  hesitate  to  say 
that  the  products  of  the  corn  kernel,  together  with  the  stem 
of  that  plant,  can  when  furnished  in  the  proper  proportions, 
serve  well  all  the  requirements  of  the  growing  and  reproducing 
heifer;  but  to  say  that  the  corn  kernel  and  its  products  can 
furnish  all  the  requirements  for  the  best  growth  and  most  vigor- 
ous reproduction  in  swine  is  to  ignore  the  results  of  many  care- 
ful experiments  furnishing  evidence  to  the  contrary. 

So  constant  by  groups  was  the  effect  of  each  ration  in  our 
experiment,  that  we  have  no  hesitancy  in  ascribing  the  influence 
to  the  feeding  materials  used.  What  particular  part  of  the 
plant  was  responsible  for  these  reactions  is  as  yet  unascertained. 

At  the  present  time  we  have  no  adequate  explanation  of  our 
results.  It  will  be  remembered  that  the  general  order  of  ex- 
cellence in  physiological  value  established  for  these  rations  was : 
com,  oats,  mixture  and  wheat.  It  should  also  be  remembered 
that  this  refers  to  the  whole  plant  and  not  to  any  part  of  the 
plant.  ^ allies  for  parts  of  the  plant  have  not  as  yet  been  estab- 
lished. Whether  the  grains  alone  would  take  the  above  order 
for  this  class  of  animals  we  do  not  as  yet  know. 

The  factors  most  likely  to  be  involved  in  these  experiments 
and  which  can  in  the  present  state  of  our  knowledge  throw 
light  on  nutritional  problems,  are  differences  in  the  proteins  of 
the  ration,  amounts  and  kinds  of  inorganic  materials  supplied, 
toxic  substances  parried  in  with  the  ration,  and  toxic  by-pro- 
ducts formed  in  the  fermentation  and  putrefaction  of  material 
in  the  tract. 

It  does  not  seem  very  probable  that  the  proteins  were  im- 


200 


Wisconsin  Experiment  Station. 


portant  factors  in  differentiating  these  feeding  materials. 
Growth  was  well  maintained  on  all  the  rations.  Had  the  pro- 
teins of  tlie  wheat  ration  been  lacking  in  certain  amino  acids 
or  polypeptide  combinations  and  the  animal  unable  to  synthe- 
size the  missing  fragments  from  other  amino  acids,  or  had 
certain  amino  acids  been  present  in  limiting  quantities,  then 
retention  of  nitrogen  for  tissue  building  would  have  been 
negative;  but  on  the  other  hand,  growth  at  least  was  con- 
stant on  all  the  sources  of  protein.  The  additional  fact  that 
the  mixed  ration,  presumably  furnishing  all  known  fragments 
of  protein  degradation,  was  not  of  standard  efficiency,  makes  it 
appear  very  possible  that  the  differences  in  physiological  value- 
of  these  rations  are  not  attributable  to  differences  in  protein 
structure. 

Nutrition  experiments  designed  to  test  the  adequacy  of  a 
single  or  limited  number  of  proteins,  and  which  have  involved 
the  animal  in  both  growth  and  reproduction,  are  very  limited. 
Rohmann  36  kept  mice  on  a mixture  of  pure  proteins  and  non- 
nitrogenous  material.  They  grew  and  reproduced  themselves. 
But  when  the  number  of  proteins  was  reduced  to  a single 
one,  reproduction  and  development  of  the  young  were  in- 
terfered with.  This  may  have  significance  for  our  experiments. 

The  relation  of  the  inorganic  side  to  the  problem  is  even 
less  definite.  Very  probably  the  acidity  to  litmus  of  the  urines* 
from  the  wheat-fed  animals  is  due  to  acid  salts,  their  for- 
mation being  dependent  upon  the  relative  supply  of  acid  and 
base  forming  radicals  in  the  feed  material.  It  is  a fact  that  the 
bases  supplied^  by  the  wheat  ration  were  lower  in  absolute 
quantity  than  in  the  corn  ration,  while  the  quantity  of  acid- 
forming radicals  supplied  by  the  two  rations  was  not  greatly 
different.  For  animals  of  this  type  the  normal  reaction  of 
the  urine  to  litmus  is  alkaline,  but  apparently  it  is  a question 
of  the  character  of  the  ration.  The  two  most  telling  facts  against 
the  direct  relation  of  the  inorganic  to  an  explanation  of  our 
results  are : inability  to  correct  the  depressing  action  of  the 
wheat  ration  by  the  use  of  alkaline  carbonates,  and  the  con- 
stancy in  the  amount  of  ammonia  in  the  urine  of  both  wheat 
and  corn-fed  animals,  indicating  the  absence  of  acidosis. 


36  Allgemeine  Med.  Central-Zeit.  1903,  No.  1;  1908,  No.  9,  cited  by 
Osborne,  Mendel,  and  Ferry,  Publication  156  Carnegie  Institution  of 
Washington. 


Effect  of  Rations  Balanced  from  Restricted  Sources.  201 


Of  course,  it  may  be  said  that  the  administration  of  alka- 
line carbonates  cannot  be  expected  to  replace  the  normal  action 
of  those  bases  natural  to  the  ration,  and  that  the  manner  of 
combination  of  the  bases  is  very  important  ; but  it  must  be 
remembered  that  experimental  data  do  show  that  inorganic 
salts  in  the  presence  of  insufficient  organic  ash,  can  serve  at  least 
the  growing  requirements  of  an  animal  for  ash  materials  and 
in  certain  instances  even  the  reproducing  requirements.  Ex- 
periments 37  from  these  laboratories  are  evidence  for  this  view. 

The  possibility  of  the  wheat  grain  carrying  poisonous  sub- 
stances, as  the  by-products  of  such  fungus  growths  as  ergot, 
was  also  raised  during  the  progress  of  this  investigation.  Ex- 
amination for  such  fungus  spores  on  samples  of  the  wheat 
used  during  the  years  1909  to  1911  were  made  by  Prof.  L.  R. 
Jones,  Plant  Pathologist  of  this  Station.  The  results  of  the 
examination  were  negative,  no  fungus  spores  developing  known 
poisons  being  reported.  As  a matter  of  fact  the  results  se- 
cured with  the  wheat  group  of  animals  were  so  slow  in  ap- 
pearance and  apparently  accumulative  in  character,  as  to  ex- 
clude, a priori,  the  presence  of  ergot  poisons. 

The  hypothesis  that  there  are  by-products  formed  from  the 
putrefaction  and  fermentation  of  certain  kinds  of  materials 
along  the  tract  is  another  possibility.  Up  to  the  present  time, 
however,  no  positive  evidence  in  this  direction  is  available. 

An  extended38  study  of  the  bacterial  flora  of  the  feces  of 
these  animals  did  not  reveal  the  presence  of  types  of  organ- 
isms peculiar  to  any  one  lot.  There  was  as  much  variation  in 
this  respect  among  the  individuals  of  a group  as  between 
groups  themselves. 

The  wheat  urines  contained  no  free  benzoic  or  hippuric  acid. 
The  content  of  hippurates  in  the  wheat  and  corn  urines  examin- 
ed was  quite  alike.  The  statement  that  none  of  the  above  acids 
were  in  a free  state  is  based  on  the  fact  that  extraction  of  the 
urine  with  acetic  ether  gave  no  free  acids.  It  should,  how- 
ever, be  stated  that  methods  for  estimating  hippuric  and  benzoic 
acids  in  urines  are  not  all  that  is  desired  and  further  work 
is  needed  on  this  phase  of  the  subject. 

There  may  be  other  aromatic  acids  present  in  the  urine,  pro- 
s’’Res.  Bui.  1,  Wis.  Exp.  Sta. 

Res.  Bui.  8,  Wis.  Exp.  Sta. 

38  Dept,  of  Agri.  Bact.  Wis.  Exp.  Sta.  (unpublished  data). 


202 


Wisconsin  Experiment  Station. 


duced  by  the  fermentation  of  certain  classes  of  materials  in 
the  paunch  or  intestinal  tract,  and  which  are  slowly  absorbed 
and  become  toxic  to  the  cells.  Also  in  this  direction  further 
efforts  to  explain  our  results  are  now  being  directed.  Toxicity 
of  some  kind,  slow  and  accumulative  in  action,  and  produced 
by  the  absorption  of  abnormal  products  of  decomposition  of 
the  straws  would  probably  best  explain  our  results.  The  lower 
plane  of  excellence  of  the  oat  ration,  than  the  corn,  and  the 
still  lower  plane  maintained  by  the  wheat  ration,  would  har- 
monize with  the  view  that  the  straws  rather  than  the  grains 
were  the  seat  of  the  trouble.  On  further  investigation,  abnor- 
mal or  excessive  quantities  of  normal  by-products  of  fermen- 
tation may  possibly  be  found  in  the  urines  of  the  wheat-fed 
mothers.  This,  at  present  is  another  working  hypothesis  and 
its  validity  will  be  thoroughly  tested. 

In  searching  for  positive  explanations  of  the  phenomena 
observed  in  these  experiments,  even  the  physical  nature  of  the 
ration  must  not  be  entirely  pushed  aside.  The  relatively  low  pro- 
duction value  of  the  straws,  as  determined  by  Kellner  and  Arms- 
by,  are  results  secured  from  their  use  over  a very  short  period 
of  time.  Including  corn  stover  the  order  of  efficiency  given 
for  the  roughage  used  in  these  experiments  was  corn  stover, 
oat  straw  and  wheat  straw.  The  possibility  that  prolonged 
feeding  of  these  materials  would  tend  to  make  the  production 
values  widely  different  from  those  established  by  experiment- 
ation over  short  periods  of  time,  is  possible  though  of  course 
remote.  The  fact  that  the  coefficients  of  digestibility  of  the 
dry  matter,  even  on  the  third  year  of  consumption  were  not 
greatly  different  for  the  different  rations,  and  that  the  rates  of 
growth  over  a long  period  of  time  were  not  materially  unlike, 
would  not  support  the  view  that  inanition  due  to  negative  pro- 
duction value  for  the  wheat  straw  was  a factor  in  our  results. 

The  question  of  measuring  vitality  or  vigor  in  a living 
organism  is  a very  important  one  in  this  class  of  experiments. 
Some  more  definite  and  accurate  way  than  the  mere  gross 
observations  of  the  offspring  or  the  individual  mother  is  need- 
ed. AVe  say  this,  however,  with  every  confidence  in  the  gen- 
eral interpretation  of  our  data  in  reference  to  both  the  mothers 
and  the  progeny  produced. 

Some  uses  of  the  catalase  reaction,  as  developed  by  Hawk39 


39  Journal  Am.  Chem.  Soc.,  vol.  33,  No.  3,  p.  425. 


Effect  of  Rations  Balanced  from  Restricted  Sources.  203 

in  his  fasting  studies,  on  the  tissues  and  organs  of  the  calves 
born  in  1911  suggest  that  such  a test  may  be  developed  into  a 
useful  instrument  for  measuring  the  catalytic  power  of  dif- 
ferent tissues  and  its  correlation  with  the  vigor  of  the  individual. 
The  conditions  for  conducting  the  reaction  are  under  further 
investigation  and  therefore  are  only  suggestive  of  greater  pos- 
sibilities. For  example,  the  kidney  tissue  from  a newly  born 
wheat  calf  showed  a power  to  decompose  hydrogen  peroxide, 
in  a given  time,  only  about  half  as  fast  as  the  same  tissue 
from  a newly  born  corn  calf.  Liver  tissue  showed  a rate  of 
about  one-third,  spleen  tissue  about  one-twentieth,  and  muscle 
tissue  about  one-fifth  as  fast,  while  heart  muscle  and  lunsr 
tissue  of  both  animals  were  apparently  alike  in  this  respect. 
Methods  for  the  measurements  of  the  concentration  and  activity 
of  other  enzymes  in  these  tissues  would  undoubtedly  be  of  great 
value  in  experiments  of  this  character. 

In  view  of  all  the  facts  presented,  it  appears  to  us  that  there 
is  a decided  physiological  value  to  our  feeds,  whose  proper  or 
improper  combination  may  make  for  vigor,  resistance  and  splen- 
did condition,  or  for  weakness,  low  resistance  and  poor  con- 
dition in  the  individual.  The  determination  of  this  physio- 
logical value,  coupled  with  the  dynamic  value  and  those  other 
essential  factors  of  adequate  ash  and  protein,  should  ultimately 
give  us  proper  scientific  ground  for  the  nutrition  of  our  do- 
mestic animals. 

Some  results  secured  in  an  early  experiment  at  this  station 
by  Dr.  S.  M.  Babcock  were  the  real  fore-runners  of  this  larger 
investigation  and  to  him  the  authors  desire  to  express  their 
great  appreciation  for  his  counsel  and  constant  interest  in  the 
development  of  this  woirk. 


Summary 

This  paper  summarizes  the  preliminary  results  of  an  ex- 
tended investigation  on  the  physiological  value  of  rations  for 
domestic  animals.  The  data  here  presented  are  limited  to  grow- 
ing and  reproducing  heifers,  and  extend  over  a period  of  four 

years. 

There  is  evidence  from  the  data  that  there  is  a distinct  and 
important  physiological  value  to  a ration  not  measureable  by 
present  chemical  methods  or  dependent  upon  mere  supply  of 
available  energy.  While  the  latter  are  important  and  give 


204 


Wisconsin  Experiment  Station. 


valuable  data  for  a starting  point,  they  are  nevertheless,  in- 
adequate as  final  criteria  of  the  nutritive  value  of  a feed. 

Animals  fed  rations  from  different  plant  sources  and  com- 
parably balanced  in  regard  to  the  supply  of  digestible  organic 
nutrients  and  production  therms  were  not  alike  in  respect  to 
general  vigor,  size  and  strength  of  offspring  and  capacity  for 
milk  secretion. 

Animals  receiving  their  nutrients  from  the  wheat  plant  were 
unable  to  perform  normally  and  with  vigor  all  the  above  physi- 
ological processes. 

Those  receiving  their  nutrients  from  the  corn  plant  were 
strong  and  vigorous,  in  splendid  condition  all  the  time,  and 
reproduced  young  of  great  weight  and  vigor. 

Animals  receiving  their  nutrients  from  the  oat  plant  wTere 
able  to  perform  all  the  physiological  processes  of  growth  re- 
production and  milk  secretion  with  a certain  degree  of  vigor, 
but  not  in  the  same  degree  as  manifested  by  the  corn-fed  ani- 
mals. 

Where  a mixture  of  all  the  above  plant  materials  was  used, 
the  animals  responded  to  the  ration  with  less  vigor  than  to  the 
corn  or  oat  rations  alone,  but  with  more  vigor  than  to  the  wheat 
ration. 

These  are  the  records  from  the  continued  use  of  rations  for 
three  years.  Monotony  of  diet  was  not  a troublesome  factor 
and  is  not  of  such  importance  in  nutrition  problems  as  usu- 
ally supposed. 

The  urines  of  the  wheat-fed  animals  were  acid  to  litmus ; 
the  urines  from  all  the  other  lots  were  alkaline  or  neutral  to 
the  same  indicator.  Correction  of  this  acid  reaction  by  feeding 
alkaline  carbonates  did  not  restore  the  wheat-fed  group  to  full 
vigor  and  proper  condition.  Allantoin  was  absent  from  the 
urines  of  this  group  during  their  period  of  growth.  During 
gestation  it  was  present.  The  degree  of  oxidation  of  sulphur 
in  the  urines  of  the  several  groups  was  not  greatly  different. 

At  present  we  have  no  solution  for  the  observations  made. 
Differences  of  protein  structure,  so  important  in  modern  the- 
ories of  nutrition,  would  appear  to  be  excluded  as  a possible 
factor  because  of  the  results  . secured  with  a mixed  ration. 
Lack  of  adequate  supply  of  bases  in  the  wheat  ration  would 
also  appear  to  be  excluded  as  a factor,  upon  the  basis  of  the 
records  secured  with  the  addition  of  inorganic  ash  mixture 


Effect  of  Rations  Balanced  from  Restricted  Sources.  205 

to  that  ration.  However,  we  reserve  for  the  future  all  final 
conclusions  as  to  the  importance  of  these  factors  to  our  results. 

Further,  the  possibility  of  toxic  bodies  being  carried  in  the 
ration,  or  of  such  substances  being  produced  in  the  tract  are 
not  too  remote  to  deserve  consideration.  Distinct  poison  pro- 
ducing fungi,  as  for  example,  ergot,  are,  however,  excluded  by 
direct  examination  of  the  wheat  grain,  as  possibilities  in  ex- 
plaining these  results. 

The  influence  of  a normal  ration,  depressing  or  stimulating, 
may  be  felt  in  a single  gestation  period.  Wheat-fed  animals 
were  changed  to  the  corn  ration  with  marked  improvement 
within  the  year  in  the  size  of  offspring  and  in  milk  secretion. 
The  converse  was  true  when  corn  animals  were  taken  to  the 
wheat  ration. 

Records  of  tissue  and  milk  composition  are  also  recorded, 
because  of  the  unusual  opportunity  of  securing  material  built 
under  the  influence  of  restricted  rations  fed  continuously  for 
such  long  periods  of  time. 


^JLAJUXrudv  WfbjMv 

A Sclerotium  Disease  of  Blue  Joint  and 
Other  Grasses1 * * * * * * 


A.  B.  STOUT 

Introduction 

During  the  summer  of  1907  the  writer  observed  a fungus 
which  was  appearing  in  the  marsh  meadows  about  Madison, 
Wis.,  as  a parasite  on  the  leaves  of  the  grass  commonly  known 
as  blue  joint  ( Calamagrostis  canadensis).  The  principal  symp- 
toms of  this  disease  a,re  as  follows.  The  portions  of  the  leaves 
attacked  soon  lose  their  green  color  and  become  dry  and  rigid. 
Often  an  entire  culm  is  killed.  When  a large  number  of  culms 
within  a small  area  are  infected,  the  general  appearance  of  the 
dead  and  whitened  leaves  is  somewhat  similar  to  that  often 
produced  on  young  grain  plants  by  a frost.  A cursory  exam- 
ination, however,  showed  that  there  was  present  on  the  dying 
leaves  a delicate  gray  felt  of  mycelium  from  which  sclerotia 
often  developed.  When  mature  these  sclerotia  project  into  the 
air  as  small  but  conspicuous  bead-like  bodies. 

These  symptoms  clearly  indicated  that  this  fungus  was  the 
eause  of  the  death  of  the  leaves  and  culms  of  the  grass  in  ques- 
tion and  the  severity  of  the  attack  made  it  a matter  of  consider- 
able economic  importance  inasmuch  as  blue  joint  is  the  most 
valuable  of  the  wild  hay  grasses  of  Wisconsin. 

Davis  (1893,  p.  183)  has  briefly  described  a fungus  which 
produces  sclerotia  on  Calamagrostis  canadensis  and  which  he 
found  at  various  points  in  Wisconsin.  He  did  not  identify 

1 The  investigations  here  reported  were  carried  on  in  part  under  the 

guidance  of  Prof.  R.  A.  Harper  of  the  department  of  Botany  of  the  Uni- 

versity of  Wisconsin.  When  Prof.  L.  R.  Jones  assumed  the  chair  of 

Plant  Pathology  at  Wisconsin  in  February  1910,  the  work  was  contin- 

ued as  a special  pathological  problem  under  his  immediate  direction. 

From  each  of  these  the  writer  has  received  helpful  criticism  and  sug- 

gestions. " 9 


208 


Wisconsin  Research  Bulletin  No.  18 


the  fungus  but  stated  that  the  sclerotia  resemble  those  which 
he  had  found  on  Silphium,  Helianthus,  etc. 

It  was  found  at  once  that  the  fungus  agrees  with  European 
specimens  of  Solerotium  rhizodes  Auersw.  growing  on  Phalaris 
arundinacea,  and  on  Calamagrostis  arundinacea.  (Sydow,  My  co- 
theca  Germanica  Nos.  298  and  299.)  Later  an  examination  of  the 
exsiccati  in  the  Ellis  collection  now  in  the  herbarium  of  the  New 
York  Botanical  Garden  showed  that  the  fungus  Sclerotium  rhi- 
zodes has  been  distributed  as  follows : 

De  Thiimen,  Fungi  Austriaci,  No.  1096,  on  Poa  pratensis, 
1873. 

De  Thiimen^  Mycotheca  Universalis,  No.  199  on  Poa  pratensis, 
1873. 

Eriksson,  Fungi  Parasitici  Scandinavici,  No.  82,  on  Poa 
pratensis  1882. 

Rabenhorst-Winter,  Fungi  Europaei,  No.  3,199,  on  Phala- 
ris arundinacea,  1884. 

Sydow,  Mycotheca  Marchica,  No.  1339,  on  Poa  leaves, 
1887 ; No.  2698,  on  Calamagrostis  negiecta,  1889 ; No.  2996, 
on  Digraphis  arundinacea,  1890 ; No.  3297,  on  Poa  trivialis, 
1891;  No.  3298,  on  Anthoxanthum  odoratum,  1891. 

Krieger,  Fungi  Saxonici,  No.  550,  on  Phalaris  anmdtina- 
cea,  1889 ; No.  600,  on  Agrostis,  1890 ; No.  1397,  on  Bracliy- 
podium  silvaticum,  1896;  No.  1398,  on  Calamagrostis  Halleriana, 
1898;  No.  1399,  on  (a)  Rolens  lanatus , 1891;  on  (b)  Rolcus 
mollis,  1897. 

Allescher  and  Schnabl,  Fungi  Bavarici,  No.  200,  on  Plialans 
arundinacea , 1891. 

The  examinations  of  these  specimens  established  beyond 
question  the  identity  of  the  Wisconsin  fungus  on  Calamagrostis 
canadensis  with  the  European  species,  and  the  writer  has  sup- 
plied specimens  so  named  for  distribution  in  Fungi  Columbiani. 

The  first  publication  and  description  of  this  fungus  was  un- 
der No.  1232  in  Klotzscli’s  Herbarium  Yivum  Mycologicum, 
a review  of  which  appeared  in  the  Botanische  Zeitung  Yol.  7, 
p.  294,  1849.  The  description  reads  as  follows  : “1232  Sclero- 
tium ( Sarcidium ) rhizodes  Awd.  Mspt.  Subglobosum,  primum 
albovillosum!,  mox  glabriusculum,  nigrescens  rugulosum,  fibrillis 
albis  seriatim  insidens.  Auf  Blattern  von  Calamagrostis  Ep-ig - 
?ios  schop  vpr  deren  Entwiekfiuig.’2 


A Sclerotium  Disease  of  Common  Grasses 


209 


Frank  (1881,  p.  545,  and  1896,  vol.  2,  p.  511)  quite  ade- 
quately describes  the  symptoms  of  this  sclerotial  disease  of 
grass  leaves.  He  records  that  the  disease  appeared  as  an  epi- 
demic in  1879  in  the  vicinity  of  Leipzig  on  Phalaris  arundinacea 
and  Dadylis  glomerata  and  that  a large  part  of  one  meadow 
appeared  dry  and  white  as  a result  of  the  attack  of  the  fungus. 
He  states  that  no  conidia  from  the  mycelium  or  fruiting  bodies 
from  the  sclerotia  had  been  observed. 

Sorauer  (1886,  p.  300)  quotes  briefly  from  Frank’s  descrip- 
tion of  1881  but  adds  no  new  data. 

Saccardo  (1899,  p.  1154)  lists  Sclerotium  rhizodes  with  the 
fungi  having  sterile  mycelia.  He  repeats  the  brief  descrip- 
tion quoted  above.  Otherwise  he  makes  no  mention  of  the  oc- 
currence of  the  fungus  as  given  in  the  various  exsiccati  listed 
above. 

Tubeuf  (1897,  p.  266)  records  this  fungus  with  sclerotia  of 
unknown  affinity  and  mentions,  only,  that  it  occurs  on  living 
plants  of  Phalaris  arundinacea , and  Calamagrostis ; also  on 
dead  leaves  of  Dadylis  glomerata. 

This  summary  of  the  literature  pertaining  to  Sclerotium 
rhizod\es  indicates  clearly  the  meagre  and  incomplete  knowl- 
edge concerning  its  life  history  and  its  relations  to  its  host 
plants. 

In  the  region  about  Madison,  Wis.,  the  fungus  is  most  abund- 
ant on  Calamagrostis  canadensis,  although  as  explained  later 
it  occurs  on  various  other  species  of  grass.  My  studies  have 
been  made  chiefly  with  material  from  this  one  host  and  all 
the  statements  which  follow  are  to  be  so  understood  unless 
otherwise  clearly  specified. 


Detailed  Description  of  Symptoms 

When  the  leaves  of  infected  plants  start  to  unfold,  their 
tips  remain  more  or  less  convolutely  rolled  as  in  the  bud  and 
soon  become  white,  dead,  dry,  and  rigid.  The  whitened  tips, 
over  badly  infected  areas,  are  conspicuous  in  mass  effect  and 
at  first  sight,  as  already  stated,  resemble  frost  injury.  Exam- 
ination, however,  reveals  the  presence  of  a thin  but  dense  felt, 
of  mycelium  which  is  most  marked  on  the  inner  surface  of  the 
infected  leaves  and  along  the  inner  edge  of  the  leaf,  especially 
when  only  a part  of  the  leaf  is  rolled  laterally.  (See  Figures 


210 


Wisconsin  Research  Bulletin  No.  18 


1 and  3,  points  marked  m.)  In  the  outermost  leaves,  the  tip 
is  usually  the  only  portion  affected  and  often  only  a lateral 
half  of  the  blade  is  invaded.  As  the  portion  of  the  leaf  at- 
tacked dies  and  fails  to  unroll,  the  tip  of  the  leaf  next  in 
order  is  often  caught  and  firmly  held  in  the  roll  and  it  in  turn 
holds  in  the  same  manner  the  leaf  next  in  order.  Meanwhile 
the  growth  of  the  basal  portion  of  the  leaves  together  with 
the  elongation  of  the  internodes  tends  to  separate  the  lower 
halves  of  the  leaves  whose  tips  are  thus  held  together  and  to 
produce  peculiar  and  characteristic  crooks  as  shown  in  Figures 
1,  2 and  3.  When  the  fungus  develops  vigorously,  the  inner 
leaves  are  completely  penetrated  by  the  mycelium  which  also 
extends  into  the  culm  below  the  growing  point.  In  this  case 
the  death  of  the  terminal  bud  results.  During  a season  of  vig- 
orous development  the  majority  of  the  infected  culms  never 
grow  to  be  more  than  twelve  inches  high,  while  large  numbers 
are  less  than  six  inches  high.  Entire  groups  of  culms  arising 
from  a rhizome  are  often  thin,  puny,  and  dwarfed. 

From  these  conditions  it  is  evident  that  the  mycelium  first 
becomes  virulent  within  the  bud  whence  it  spreads  through 
leaf  after  leaf  and  that  the  individual  leaves  are  thus  infected 
before  they  unroll  from  the  bud.  The  mycelium  becomes  most 
conspicuous  on  a leaf  in  the  lowest  part  of  its  region  of  growth. 
The  unaffected  basal  part  of  the  blade  becomes  flattened  out 
and  just  below  the  point  where  the  next  leaf  in  order  emerges 
from  the  roll  an  area  is  usually  covered  with  a felt  of  white 
mycelium. 

In  case  of  partial  lateral  infection  a narrow  zone  with  tufts 
and  knots  of  mycelium  appears  along  the  margin  of  the  fold 
or  roll  where  the  dead  leaf  tissue  meets  the  green  tissue.  Soon 
rounded  bead-like  sclerotia  are  produced  along  the  infected  por- 
tions of  the  leaf  or  from  the  felt  of  mycelium  at  the  base.  (See 
Figure  1 points  marked  s).  When  mature,  they  vary  in  size 
from  1 to  5 mm.  in  diameter  (See  Figure  3).  The  sclerotia 
are  formed  on  the  leaves  and  are  always  superficial.  They  are 
seldom  formed  within  the  roll  and  never  within  the  tissues. 

The  production  of  the  conspicuous  crooks  and  the  develop- 
ment of  the  sclerotia  as  described,  are  features  which  make 
certain  the  identification  of  this  'fungus  as  it  appears  on  Cala- 
magrostis  canadensis.  The  writer  has  found  this  fungus  ap- 
pearing in  the  region  about  Madison  on  the  following  addi- 


A Sclerotium  Disease  of  Common  Grasses 


211 


tional  grasses:  Phalaris  arundinacea  L. ; Calamagrostis  reglecta 
(Ehrh.)  Gaertn;  Poa  pratensis  L. ; Panicularia  nervata 
(Willd.)  Kuntze;  PJileum  pralense  L. ; Hordeum  jubatum  L. ; 
Bromus  ciliatus  L. ; Eatonia  Pennsylvania  (DC.)  A.  Gray; 
Agropyron  caninum  (L.)  R.  & S. ; Agrostis  hyemalis  (Walt.) 
B.  S.  P.  All  of  these  except  the  first  three  named  are  here  re- 
ported as  hosts  for  the  first  time.  The  occurrence  of  the  fun- 
gus upon  several  European  grass  hosts  not  here  mentioned  has 
already  been  noted.  In  general  the  appearance  of  the  fungus 
on  all  of  these  hosts  is  similar  to  that  described  fo,r  Calamagros- 
tis canadensis.  In  each  case  the  sclerotia  produced  are  identical 
and  infected  plants  of  each  species  have  been  found  growing 
by  the  side  of  infected  plants  of  Calamagrostis  canadensis. 

It  should  be  noted  that  in  the  case  of  perennial  grasses  a bud 
which  grows  upward  into  the  air  produces  leaves  only,  for 
one  or  more  years  and  then  terminates  its  life  as  an  individual 
culm  by  producing  flowers  and  seed.  . There  is  considerable 
difference  in  the  habit  of  growth  of  these  leaf  and  fruiting 
culms  in  the  different  species  that  serve  as  host  plants.  In  Poa 
pratensis  and  Panicularia  nervata  the'  vegetative  culms,  as  I 
shall  designate  the  culms  bearing  leaves  only,  are  short  and  the 
leaves  which  they  bear  arise  rather  close  to  the  ground.  Here 
the  infected  leaves  are  not  lifted  up  to  the  general  level  of  the 
vegetation  and  although  there  may  be  many  infected  leaf  culms, 
the  general  effect  is  not  so  conspicuous  as  it  is  in  the  case  of 
Calamagrostis  canadensis.  The  leaves  of  these  two  species  are 
normally  conduplicate  in  thje  bud  and  the  infected  leaves  re- 
main thus  folded.  Sclerotia  a,re  produced  between  the  folds  on 
the  upper  surface  and  are  always  superficial.  Along  the  groove 
of  the  fold,  a thin  felt  of  mycelia  develops.  On  Poa  prate?i- 
sis  especially,  the  distribution  of  the  fungus  is  rather  irregular 
and  not  continuous  in  a single  leaf. 

During  the  seasons  of  1907,  1908  and  1909,  general  observa- 
tions were  made  in  the  ma,rsh  meadows  about  Madison  as  to 
the  abundance  and  the  course  of  development  of  the  fungus. 
In  1910  and  1911  however,  a single  marsh  meadow  conveniently 
situated  for  observation  was  selected  for  a more  intensive  study 
of  these  problems.  This  meadow  is  almost  circular  in  shape 
with  a diameter  of  nearly  half  a mile.  Although  its  elevation 
is  but  a few  feet  above  the  level  of  Lake  Monona,  which  is  near 
by  and  into  which  it  is  drained,  it  is  usually  sufficiently  dry  to 


212 


Wisconsin  Research  Bulletin  No.  18 


be  cut  over  for  hay.  The  statistical  analysis  .which  the  writer 
made  of  the  plant  population  of  this  marsh  meadow 
showed  that  Calamagrostis  canadensis  was  quite  generally  dis- 
tributed over  the  marsh  and  that  it  constituted  18  per  cent  of 
the  entire  plant  population.  The  counts  were  made  rather  late 
in  a season  in  which  the  fungus  was  not  especially  vigorous  ex- 
cept in  certain  areas  through  which  the  transect  studied  did  not 
pass.  The  fungus  was  noticed  at  nearly  all  points,  but  no 
separate  count  was  made  of  the  infected  culms. 

Seasonal  Development  in  the  Field  The  development  of 
the  fungus  was  further  studied  in  the  field  in  the  spring  of 
1910.  It  was  an  early  spring.  On  March  24,  the  first  grass 
buds  were  beginning  to  unfold  and  many  of  these  showed  the 
typical  effects  of  the  fungus.  Sclerotia  which  had  been  formed 
during  the  previous  year  were  found  in  numbers  on  the  ground 
but  none  of  them  showed  any  signs  of  producing  fructifica- 
tions. 

On  April  15,  the  grass  stood  from  four  to  six  inches  high. 
In  certain  areas  where  infection  was  most  general  in  previous 
years,  the  fungus  was  especially  abundant.  In  some  areas  of 
several  square  rods  extent,  it  appeared  that  75  per  cent  of  the 
culms  then  unfolding  their  buds  were  infected. 

On  April  30,  the  grass  averaged  one  foot  high.  The  dead 
tips  gave  to  the  regions  of  worst  infection  a conspicuous  whit- 
ened appearance  as  if  the  tips  had  been  burned  or  frozen. 
Many  of  the  infected  culms  were  totally  dead.  Young  sclero- 
tia were  forming  in  considerable  number. 

Throughout  May  and  June  there  was  no  apparent  increase 
in  the  number  of  culms  showing  the  infection.  The  majority 
of  culms  already  diseased  had  died  to  the  ground.  Others  con- 
tinued to  grow  and  put  forth  new  leaves  which  in  turn  showed 
the  presence  of  the  fungus  in  varying  degrees  of  vigor.  The 
season  was  one  of  unusual  dryness  and,  as  a result,  few  sclero- 
tia were  produced.  By  July  14  there  were  few  areas  where 
the  fungus  appeared  abundantly  on  the  growing  culms.  In 
areas  of  previous  slight  infection,  healthy  culms  now  stood 
about  two  feet  tall  overtopping  the  dead  culms  killed  earlier  in 
the  season.  Still  during  the  remainder  of  the  season,  culms 
bearing  infected  leaves  could  always  be  found  in  considerable 
number.  The  grass  itself  made  little  growth  after  the  middle 
of  July  and  during  the  first  week  in  August  it  was  cut  for  hay. 


A Sclerotium  Disease  of  Common  Grasses 


213 


On  September  2,  the  new  growth  was  nearly  one  foot  high 
and  in  this,  infected  culms  were  scarce  and  the  characteristic 
crooks  were  not  well  developed. 

Observations  in  1911  The  spring  of  1911  was  nearly  three 
weeks  later  than  that  of  1910.  The  fungus  appeared  as  in  pre- 
vious years  on  the  first  leaves  that  opened  and  the  subsequent 
development  was  as  described  above.  There  was  a fair  amount 
of  rainfall  through  April  and  May  and  by  May  27,  it  was  evi- 
dent from  the  number  of  affected  culms  that  the  infection  was 
more  vigorous  and  more  general  than  in  previous  seasons.  Scler- 
otia  were  abundant  and  many  were  mature  on  May  27. 

Throughout  June  the  dead  and  whitened  tips  of  infected 
grass  blades  were  abundant  and  conspicuous  especially  on  culms 
which  continued  to  develop  new  leaves.  There  was,  however, 
no  increase  as  the  season  advanced  in  the  number  of  infected 
culms.  Areas  which  were  free  from  the  fungus  earlier  in  the 
season  remained  free  from  it.  These  observations  made  it  clear 
that  there  is  no  spread  of  the  fungus  from  culm  to  culm  aerially 
and  that  a culm  which  does  not  show  the  fungus  on  its  first 
leaves  does  not  harbor  it  later  in  the  season. 

The  effect  of  the  fungus  is  especially  marked  on  Calamagros- 
tis  canadensis.  Many  of  the  culms  die  to  the  ground  early  in 
the  season.  The  remainder  continue  to  put  forth  infected  leaves, 
but  are  decidedly  stunted.  Very  few  infected  culms  produce 
flowers.  Even  apparently  uninfected  culms  which  arise  from 
the  same  rhizomes  with  infected  culms,  are  weak  and  stunted. 
Observations  made  year  after  year  show  a decrease  in  the  num- 
ber of  plants  of  C.  canadensis  in  the  infected  areas.  Throughout 
the  greater  pa,rt  of  the  marshes  C.  canadensis  is  associated  with 
Carex  stricta  with  which  it  is  more  or  less  in  competition.  The 
destructive  effects  of  the  fungus  seem  to  be  an  important  factor 
in  favor  of  Carex  stricta. 

Observations  made  each  year  indicate  that  the  fungus  devel- 
ops during  the  spring  and  early  summer.  It  is  most  vigorous 
and  conspicuous  during  the  period  of  the  most  rapid  growth  of 
the  host  plant.  It  becomes  less  noticeable  as  the  season  ad- 
vances due  to  the  death  of  many  culms  and  to  the  overtopping 
by  unaffected  culms  and  by  associated  vegetation.  The  fungus 
seems  unable  to  make  headway  on  fully  developed  leaves  during 
June  and  July.  Each  leaf  as  it  unfolds  contains  the  fungus,  but 


214 


Wisconsin  Research  Bulletin  No.  18 


the  exposure  to  dry  and  heated  air  checks  the  development  of 
mycelium  and  sclerotia. 

Abundance  and  Distribution 

To  ascertain  the  amount  of  infection  the  following  method 
was  used  upon  the  marsh  selected  for  critical  study.  At  inter- 
vals of  fifteen  paces  along  a line  leading  through  the  marsh  .a 
heavy  wire  hoop  twelve  inches  in  diameter  was  dropped  down 
at  random.  All  the  vegetation  inside  the  hoop  was  then  cut 
close  to  the  ground,  the  infected  and  the  uninfected  culms  of 
C.  canadensis  were  sorted  out  and  counted  and  the  results 
tabulated.  The  greatest  injury  appeared  to  be  at  the  borders 
of  the  marsh  meadow,  especially  on  an  area  of  about  sixteen 
acres,  which  is  somewhat  isolated  from  the  greater  part  of  the 
marsh  by  a railroad  embankment  and  a canal  which  passes 
through  the  marsh.  This  area  is  well  drained  and  is  always  dry 
enough  to  be  mowed  by  machine.  Here  the  dominant  species 


Table  I Distribution  of  Fungus  on  Galamagrostis  canadensis 


Station 

Number  of 
infected 
culms 

Healthy 

culms 

1 

1 Q 

Jo 

19 

6 

4 

4 

21 

17 

6 

J i 

A O 

7 

OQ 

8 

Q7 

26 

Ot 

1 A 

31 

14: 

APi 

5 

4D 

8 

OO 

91 

7 

32 

Do 

1 Q 

29 

1 0 
A 

6 

V 

A 

y 

•y 

u 

7 

10 

18 

6 

Total 

426 

91  £ 

*1D 

are  Car  ex  stricta,  Calamagrostis  canadensis,  Poa  prater  sis, 
Glyceria  nervata  and  Spartina  cynosuroides.  These  are  consid- 
erably intermingled.  Poa  pratensis  is  especially  abundant 
along  the  edges  near  the  upland.  C.  canadensis  is  quite  uni- 
formly distributed  over  the  area  except  at  the  extreme  bor- 
der. Beginning  at  the  margin,  data  on  the  distribution  of  the 
fungus  were  taken  as  described  above  at  seventeen  consecutive 
points  fifteen  paces  apart.  The  results  for  C.  canadensis  are 
given  in  Table  I. 


A Sclerotium  Disease  of  Common  Grasses 


215 


From  these  data  it  appears  that  66  per  cent  of  the  culms  of 
G.  canadensis  growing  on  this  area  were  infected.  While  the 
fungus  was  quite  general  in  its  distribution  on  this  area  there 
were  patches  of  small  extent,  usually  from  one  to  two  .rods  in 
diameter,  that  were  almost  entirely  free  from  the  fungus.  Often 
these  would  be  entirely  surrounded  by  infected  strips  and  in 
such  cases  the  contrast  was  always  marked.  Data  taken  from 
typical  areas  show  that  on  May  27  the  healthy  plants  then  stood 
on  the  average,  30  inches  high,  while  the  tallest  of  the  infected 
ones  nearby  were  but  12  inches,  and  the  majority  of  them  were 
4 to  8 inches  high.  Even  the  culms  that  had  escaped  infection 
were  smaller  and  poorly  developed.  These  “islands”  of  unin- 
fected plants  in  the  otherwise  destructively  infected  regions 
gave  by  comparison  conspicuous  evidence  of  the  damage  done 
by  the  disease. 

Data  on  the  abundance  of  the  fungus  over  the  greater  part 
of  the  marshes  given  in  Table  II  were  obtained  in  the  same 


Table  II  Additional  Data  on  Distribution  of  Fungus  on 
Calamagrostis  canadensis 


Station 


1 

2 

3 

4 

5 

6 

7 

8 
9 

10 

11 

12 

13 

14 

15 

16 

17 

18 

Totals  for  the  37 
stations 


I Infected 
culms 

Healthy 

culms 

Station 

Infected 
1 culms 

] 

Healthy 

culms 

0 

0 

19 

13 

24 

0 

9 

20 

5 

12 

6 

3 

21 

0 

6 

2 

5 

22 

0 

13 

7 

7 

23 

0 

0 

2 

18 

24 

0 

0 

0 

0 

25 

0 

4 

14 

7 

26 

3 

12 

6 

10 

27 

6 

19 

l 

2 

28 

5 

28 

8 

22 

29 

0 

0 

1 

37 

30 

0 

0 

14 

15 

31 

1 

12 

17 

6 

32 

1 

10 

7 

2 

33 

0 

19 

8 

27 

34 

0 

8 

27 

21 

35 

6 

24 

8 

45 

36 

19 

22 

37 

4 

8 

Aa<7 

J uO 

manner.  The  transect  began  at  the  south  edge  and  extended 
through  the  center  of  the  marsh  as  far  as  the  canal  and  thus 
coincided  with  the  transect  previously  studied  in  determining 
the  plant  population  of  this  meadow. 

For  this  part  of  the  marsh  the  infection  averaged  29  per 
cent.  Combining  all  the  data  obtained  gives  an  average  infec- 
tion of  47  per  cent  for  the  two  transects  which  represents  fairly 


Wisconsin  Research  Bulletin  No.  18 


216 

the  conditions  on  this  particular  meadow  on  May  27,  1911. 
One  transect  passed  through  a well  drained  marginal  area.  The 
other  passed  from  the  margin  through  the  wettest  part  of  the 
marsh  meadow.  The  two  give  a fair  average  of  the  whole 
meadow.  1 . : j 

In  the  central  wet  portions  of  the  meadow  Carex  dquatilis, 
Carex  Sartwellii  and  Carex  filiformis  were  dominant  and  C. 
canadensis  was  either  absent  or  sparse.  It  is  noticeable  that  the 
percentage  of  infected  culms  was  relatively  lower  under  such 
conditions.  (See  data  for  stations  20-34  in  Table  II.)  The 
sparseness  of  the  grass  evidently  gives  less  opportunity  for  the 
infection  to  spread  through  the  soil. 

Throughout  the  entire  marsh  there  were  areas  usually  of  small 
extent  that  were  free  from  infection.  There  were  also  areas 
with  more  than  90  per  cent  of  infection.  The  latter  were  uni- 
formly located  in  areas  where  C.  canadensis  was  dominant. 

Range  of  the  Disease  in  Wisconsin  During  June  1911,  the 
writer  spent  three  days  in  making  observations  on  the  occur- 
rence of  this  fungus  in  the  extensive  marshes  in  the  townships 
of  Albion  and  Christiana,  Dane  county,  Wis.  Here  several 
square  miles  of  marsh  meadow  were  traversed  extending  for 
nearly  eight  miles  along  Saunders ’ C,reek.  These  meadows 
have  for  a number  of  years  either  been  cut  for  hay  or  utilized 
as  pasture.  C.  canadensis  and  Carex  stricta  were  dominant  over 
most  of  the  area.  The  former  grew  in  luxuriance  over  large 
areas  and  stood  when  in  blossom  4 to  5 feet  high.  Carex  aqua- 
tilis  and  Carex  riparia  were  abundant  in  the  wetter  parts  of  the 
area  and  Poa  pratensis,  Panicidaria  nervata  and  Poa  flava  were 
common  near  the  borders.  In  several  areas  Phleum  pratense 
was  abundant. 

Over  the  entire  area  visited,  the  fungus  was  found  to  be  con- 
spicuously abundant  on  C.  canadensis,  although  areas  of  several 
acres  were  found  which  were  nearly  free  from  the  fungus.  On 
others  the  fungus  although  abundant  was  much  scattered  and 
its  effects  not  conspicuous.  Over  larger  areas,  however,  the 
same  degree  of  destruction  was  seen  as  has  been  described  for 
the  vicinity  of  Madison.  For  the  season  of  1911  the  infection  on 
this  entire  area  was  not  less  than  10  per  cent  of  the  culms  of 
C.  canadensis.  Besides  this  there  was  a less  conspicuous  and 
less  general  injury  to  various  other  grasses  by  the  same  fun- 
gus. 


A Sclerotium  Disease  of  Common  Grasses 


217 


In  this  region  several  small  isolated  patches  of  C.  canadensis , 
located  on  high  land,  were  found  to  be  infected.  In  fact  the  most 
uniform  and  complete  destruction  seen  anywhere  was  on  such 
an  area.  A nearly  pure  formation  of  this  species  measuring 
2%x2  rods  was  found  by  the  roadside  bordering  a cultivated 
field  and  on  high  dry  land  with  the  nearest  marsh  three-fourths 
of  a mile  distant.  The  undisturbed  dead  culms  of  previous 
years  formed  a rich  mulch.  Except  for  a fringe  at  the  ends  of 
this  formation,  practically  every  culm  was  infected.  Many 
were  entirely  dead  and  the  short  culms  with  the  uniformly 
white  tips  appeared  in  sharp  contrast  to  the  surrounding  green 
vegetation.  The  infected  belt  extended  to  the  border  of  an  oat 
field,  but  no  trace  of  the  fungus  was  found  on  the  oat  plants. 

The  occurrence  of  the  fungus  in  such  isolated  areas  of  Cal- 
amagrostis  suggests  either  that  the  fungus  is  a widespread  soil 
or  root  fungus  which  does  not  always  show  parasitic  develop- 
ment in  the  leaves,  or  that  it  is  distributed  by  spores  which 
cause  a rapid  infection  of  roots,  stems,  and  leaves.  As  noted 
above  I have  so  far  found  no  spore  stage. 

Marshes  were  also  visited  in  the  region  about  Fort  Atkinson 
and  Lake  Koshkonong  in  Jefferson  county,  Wis.  In  several 
small  isolated  marshes,  no  trace  of  the  fungus  was  found,  but 
in  long  stretches  of  marsh  land  along  Bark  River  and  Rock 
River,  the  fungus  was  abundant.  Here  again  it  was  often 
found  in  roadside  patches  of  C.  canadensis  on  rather  high  land. 
The  most  general  occurrence  of  the  fungus  found  anywhere 
was  near  the  mouth  of  Koshkonong  Creek.  Here  a continuous 
marsh  meadow  of  eighty  acres  was  examined,  June  23.  A heavy 
growth  chiefly  of  red  top  and  Car  ex  strict  a covered  the  higher 
portions  while  Phalaris  arundinacea  stood  five  feet  tall  over  the 
lower  parts.  C.  canadensis  was  abundant  over  areas  in  which 
nearly  every  culm  was  infected  with  the  fungus  so  that  the  en- 
tire Calamagrostis  population  was  overtopped  by  Carex  stricta, 
Agrostis  alba  or  other  plants  usually  of  lower  stature.  The  in- 
fection wa&  so  complete  that  not  a single  flowering  culm  of  C. 
canadensis  was  observed  on  the  entire  eighty  acres.  Phalaris 
arundinacea  was,  however,  but  slightly  affected  with  the  fun- 
gus. 

From  various  reports  it  seems  that  these  conditions  prevail 
throughput  the  greater  part  of  the  state.  Dr.  J.  J.  Davis  states 


218 


"Wisconsin  Research  Bulletin  No.  18 


in  a letter  to  the  writer:  “I  first  noticed  Sclerotium  rhizodes  on 
C.  canadensis  in  1892  and  I have  seen  it  on  that  host  every  year 
that  I have  been  in  the  field  since.  I have  collected  it  at  both 
ends  of  the  longest  axis  of  the  state  and  at  various  intermediate 
points  so  that  I think  that  it  may  be  said  to  be  generally  distri- 
buted through  Wisconsin  and  to  be  a constant  and  general  mem- 
ber of  the  parasitic  fungus  flora  of  the  state.  ’ ’ 

In  response  to  inquiries,  George  L.  Peltier  of  the  State  Cran- 
berry Experiment  Station  at  Grand  Rapids,  Wis.,  writes  as 
follows:  “I  have  made  observations  on  Sclerotium  rhizodes,  but 
have  been  able  to  find  it  only  on  blue  joint  (C.  canadensis.)  It 
is  very  widespread  here  and  I have  found  it  wherever  I have 
looked  for  it.  In  a field  just  west  of  the  station  about  30  per 
cent  of  the  stalks  seemed  affected.  On  one  of  my  trips  I found 
a whole  field  of  many  acres  where  almost  every  plant  was  af- 
fected. It  had  weakened  the  grass  so  mtich  that  none  was  able 
to  head  out.” 

It  appears,  however,  that  this  fungus  has  not  been  reported 
in  America  outside  of  Wisconsin.  This  is  most  singular.  Here 
in  Wisconsin  it  is  widespread,  abundant  and  conspicuously 
parasitic.  Its  chief  host,  C.  canadensis  ranges  from  New- 
foundland to  Alaska  south  to  North  Carolina,  New  Mexico 
and  California  and  other  grasses  which  may  serve  as  hosts  in- 
crease the  area  in  which  the  fungus  may  appear.  It  seems  prob- 
able that  this  fungus  is  equally  common  in  other  sections  besides 
Wisconsin,  but  has  been  overlooked. 

Extent  of  Infection  on  Grasses  Other  Than  Calamagrostis 

Canadensis 

The  frequency  of  the  fungus  on  other  grasses  bears  directly 
on  the  question  as  to  the  method  of  infection. 

Phalaris  arundinacea  Scattering  groups  of  plants  were 
found  with  the  fungus  in  the  marsh  meadows  along  Albion 
Creek,  Rock  River  near'  Ft.  Atkinson,  and  Lake  Koshkonong. 
These  were  always  in  the  immediate  vicinity  of  badly  infected 
areas  of  C.  canad\ensis  and  wTere  usually  at  the  border  of  a Pha- 
laris formation.  On  the  whole,  this  species  was  slightly  infect- 
ed in  this  region.  This  grass  did  not  occur  in  the  marsh  mea- 
dows studied  at  Madison, 


A Sclerotium  Disease  of  Common  Grasses 


219 


Calamagrostis  neglecta  The  fungus  was  abundant  and  in- 
jurious over  areas  covered  with  this  species.  Infected  C.  cana- 
densis was  always  found  in  the  vicinity  and  usually  the  two 
species  were  intermingled. 

Panicularia  nervata  Wherever  this  species  grew  inter- 
mingled with  infected  C.  canadensis  a small  per  cent  of  its  leaf 
culms  showed  infection.  On  this  host  however,  the  fungus  did 
not  appear  to  be  seriously  destructive. 

Poa  pratensis  In  the  case  of  this  species  there  was  rather 
abundant  and  serious  injury  especially  where  infection  of  C. 
canadensis  was  vigorous  in  the  immediate  vicinity,  but  the  fun- 
gus also  appeared  quite  abundantly  in  the  nearly  pure  forma- 
tions of  Poa  pratensis , which  thrive  in  the  border  and  upland 
portions  of  marshes  about  Madison. 

Phleum  pratense  Vigorous  infection  of  this  grass  was  seen 
in  the  border  of  a marsh  near  Albion.  Only  a few  infected 
leaves  and  culms  were  found  and  in  nearly  every  case  these  grew 
by  the  side  of  infected  culms  of  C.  canadensis. 

Hordeum  jubatum  At  Madison  this  grass  was  found  grow- 
ing in  border  areas  of  the  marsh  often  with  its  roots  inter- 
mingled with  those  of  infected  culms  of  C.  canadensis.  It  was 
only  under  these  conditions  that  culms  were  observed  showing 
the  characteristic  effects  of  the  fungus. 

Bromus  ciliatus,  Eatonia  pennsylvanica,  Agrostis  hyemalis, 
Agropyron  caninum.  A few  culms  of  each  of  these  species  were 
found  infected  with  the  fungus.  Infected  plants  of  C.  cana- 
densis were  always  close  at  hand. 

These  observations  suggest  that  while  C.  canadensis  serves  as 
the  principal  host  for  Sclerotium  rhizodes  the  fungus  may  spread 
to  various  other  grasses  especially  when  they  are  in  close  prox- 
imity, a fact  which  is  fully  explained  when  the  soil  and  root 
relationships  of  the  host  are  considered.  Several  species  of 
grasses,  especially  Agrostis  alba , Andropogon  furcatus  and 
Sp.artina  cynosuroides , appear  to  be  immune,  or  at  least  no  evid- 
ences of  the  fungus  were  found  on  plants  of  these  species  which 
grew  within  zones  of  infected  C.  canadensis. 

It  is  of  interest  to  note  that  on  the  stems  of  Urtica  gracilis , 
which  was  growing  within  an  area  of  vigorous  development  of 
the  fungus  on  C.  canadensis,  there  we.re  found  in  one  season 
sclerotia  somewhat  similar  to  those  on  C.  canadensis. 


220 


Wisconsin  Research  Bulletin  No.  18 


Conditions  Favorable  for  Development 

It  has  already  been  shown  that  the  fungus  usually  developed 
most  abundantly  in  the  field  during  the  early  part  of  the  season, 
but  that  during  moist  summers  it  was  also  vigorous  later  in  the 
season.  To  determine  the  conditions  which  favor  the  develop- 
ment of  the  aerial  mycelium  and  the  production  of  sclerotia  the 
following  studies  were  made  during  April  and  May  1910. 

Culms  in  the  early  stages  of  development  were  gathered  in  the 
field,  immediately  placed  in  sterile  test  tubes  about  4x20  cm. 
in  size  and  plugged  about  the  stem  with  cotton.  These  were 
taken  to  the  greenhouse  and  so  placed  that  the  cut  end  of  the 
culm  and  the  mouth  of  the  test  tube  were  in  water  thus  forming 
a damp  chamber  of  the  test  tube.  Clumps  of  the  plants  with 
infected  culms  were  also  transplanted  into  pots  and  kept  under 
bell  jars.  For  comparison  others  were  exposed  to  the  air  of  the 
greenhouse.  In  all  the  latter  the  fungus  developed  slowly  with- 
out any  conspicuous  show  of  mycelium  and  the  sclerotia  began 
to  form  in  about  ten  days.  Here  the  development  and  appear- 
ance closely  resembled  that  observed  in  the  field. 

In  the  case  of  the  infected  culms  placed  in  sterile  test  tubes 
there  appeared  within  twenty-four  hours  an  abundant  mycelial 
growth  which  extended  from  the  infected  portions  of  the  leaves 
out  into  the  tube  forming  a cottony  mass  which  often  filled  the 
tube  for  one-half  or  two-thirds  its  length.  Soon  numerous  scle- 
rotia began  to  form.  Many  of  these  were  out  in  the  aerial  my- 
celium and  were  not  directly  attached  to  the  leaves.  At  the 
end  of  fifteen  days  the  culms  and  unaffected  portions  of  the 
leaves  were  still  green,  the  mycelium  was  still  vigorous  and 
many  of  the  sclerotia  we, re  fully  mature. 

There  was  a less  vigorous  development  of  aerial  mycelium  on 
potted  plants  inclosed  in  a bell  jar.  Many  infected  culms  died 
in  ten  to  twenty-four  days  while  noninfected  culms  remained 
green.  Sclerotia  on  these  plants  were  mature  in  about  twenty 
days.  These  experiments  show  that  increased  humidity  favors 
the  development  of  the  mycelium  on  the  surface  of  the  leaves 
and  promotes  rapid  formation  of  the  sclerotia,  wdrich  agrees  with 
the  facts  observed  in  the  field  as  previously  discussed. 


221 


A Sclerotium  Disease  of  Common  Grasses 


Source  of  Infection 

During  the  past  three  years  hundreds  of  infected  culms  have 
been  examined  in  all  stages  of  the  disease  and  throughout  the 
entire  period  of  its  appearance,  but  no  spores  were  found.  Evid- 
ently the  mycelium  is  prevailingly  sterile  as  it  occurs  in  nature. 

The  history  of  many  other  sclerotia-forming  fungi  suggests 
that  the  sclerotia  may  develop  aseoca.rps  with  ascospores.  Dil- 
igent search  for  germinating  sclerotia  has  been  made  during 
each  of  the  past  three  years.  Each  year  as  soon  as  the  snow 
melted  sclerotia  were  found  on  the  ground  in  the  areas  of  worst 
infection  and  they  were  found  and  examined  in  situ  throughout 
the  season.  No  evidence  of  germination  was  found. 

Both  sclerotia  gathered  from  the  grass  in  the  field  and  those 
grown  in  cultures  have  been  treated  in  a variety  of  ways  in  the 
endeavor  to  induce  germination,  but  with  no  success  at  present 
writing.  During  the  season  of  1910  almost  no  sclerotia  were 
matured  in  the  field,  but  in  1911  they  were  produced  in  abund- 
ance and  I have  at  least  500  now  planted  under  a variety  of 
conditions.  In  the  case  of  a few  sclerotia  gathered  from  Urtica, 
germination  was  secured,  but  a discussion  of  these  is  reserved 
until  the  identity  of  the  fungus  is  more  certain. 

So  far  as  I can  find  the  sclerotia  do  not  germinate  freely 
and  abundantly  each  year.  They  are,  in  fact,  not  matured 
in  abundance  each  year,  for  by  fa,r  the  greater  number  dry 
up  and  become  shriveled,  while  immature.  Yet  the  fungus  is 
abundant  year  after  year.  It  was  noted  that  the  infection  re- 
appeared year  after  year  in  the  same  areas  and  that  patches 
near  by  were  constantly  free  from  infection.  Examination  also 
showed  that  in  many  cases  the  majority  if  not  all  of  the  culms 
arising  from  the  same  .root  stalk  were  infected.  The  primary  de- 
velopment in  every  case  seemed  to  be  from  within  the  bud  for 
here  the  fungus  appears  when  the  first  leaves  unfold.  All  this 
evidence  suggested  that  the  mycelium  may  be  perennial.  To 
test  this  the  following  experiment  was  made. 

March  5,  1910,  while  the  snow  stood  nearly  two  feet  deep  on 
the  marsh,  rhizomes  of  the  Calamagrostis  were  dug  from  an 
area  where  the  fungus  had  been  abundant  during  the  previous 
season,  taken  to  the  greenhouse  and  potted  in  muck  soil  that  had 
been  in  use  in  the  greenhouse  for  some  fifteen  years.  In  the 
potting,  the  old  culms  and  dead  leaves  were  removed  so  that 


222 


Wisconsin  Research  Bulletin  No.  18 


the  rhizomes  and  buds  only  were  planted.  The  soil  was  kept 
well  watered.  March  14  the  young  culms  were  from  two  to 
four  inches  tall  and  in  one  of  the  opening  buds  the  mycelium 
of  the  fungus  was  visible.  March  25  this  culm  showed  three 
leaves  infected  with  the  fungus  and  producing  the  typical 
crooks.  At  this  date  the  opening  leaves  of  three  other  culms 
showed  the  presence  of  the  fungus,  two  culms  were  dead  from 
the  effects  of  the  fungus,  and  eight  were  apparently  free  from 
infection.  The  culms  were  12  to  15  inches  tall  on  April  1,  with 
as  many  as  five  leaves.  The  infected  culms  showed  the  typical 
development  seen  in  the  field2.  These  results  are  quite  conclu- 
sive that  the  mycelium  is  present  in  the  buds  during  the  winter. 

Relation  of  the  Host  and  the  Fungus 

Method  of  Investigation  Following  the  above  experiment 
the  infected  a,reas  were  visited,  and  leaves,  buds,  portions  of 
stems  and  rhizomes  of  infected  plants  were  fixed  in  chrom-acetic 
and  picro-formal  fixing  solutions.  This  material  was  imbedded, 
sectioned  and  stained  with  either  iron-haematoxylin  or  with  the 
Fleming  triple  stain.  The  sections  showed  that  the  mycelium 
is  coexistent  in  and  on  the  leaves,  buds,  stems,  rhizomes,  and 
roots  of  the  same  plants.  The  characteristics  of  the  fungus  in 
these  different  parts  are  such  that  a detailed  description  of  each 
is  necessary. 

Character  of  the  Aerial  Mycelium  On  the  matured  foliage 
leaves,  the  mycelium  is  in  part  aerial  as  described  above.  The 
mycelium  is  white  in  mass.  It  is  abundantly  branched,  is  sep- 
tate, and  the  hyphae  anastomose  to  some  extent.  The  ends  of 
the  hyphae  are  often  enlarged.  The  walls  are  thin  and  the  cyto- 
plasm is  slightly  granular  and  much  vacuolated.  Figure  8 B 
shows  the  general  appearance  of  the  aerial  mycelium  taken 
from  the  surface  of  the  leaf. 

For  a more  careful  study  of  the  cell  structure,  the  mats  of 
mycelium  produced  in  cultures  on  cooked  potato  were  fixed  in 


2 This  experiment  has  been  repeated  with  the  following  data:  No- 

vember 24,  1911,  Mr.  A.  G.  Johnson  chopped  from  the  frozen  ground 
at  Madison  rhizomes  of  Calamagrostis  canadensis  and  Poa  pratensis . 
These  were  sent  to  me  at  the  New  York  Botanical  Garden  and  im- 
mediately placed  in  pots  which  were  kept  in  a greenhouse.  As  soon 
as  the  first  leaves  of  the  growing  culms  unfolded  (January  8,  1912) 
the  fungus  was  found  with  typical  development  in  the  buds  of  several 
culms  of  both  species. 


A Sclerotium  Disease  of  Common  Grasses 


223 


Fleming’s  weak  solution  and  stained  by  either  the  triple  method 
o,r  by  iron  haematoxylin.  This  treatment  showed  that  some  of 
the  cells  of  the  mycelium  were  two-nucleated  with  a reticulated 
protoplasm  as  shown  in  Figure  7,  J.  I have  not  found  division 
figures  in  any  cells  of  the  fungus  and  I can  give  no  data  on  the 
constancy  of  the  two-nucleated  condition  or  its  possible  origin 
in  conjugate  division. 

A few  cells,  however,  had  more  than  two  nuclei  and  occasion- 
ally only  one  nucleus  was  present.  The  general  character  of 
the  mycelium  differed  on  the  various  culture  media.  This  will 
be  described  later. 

The  Mycelium  in  the  Leaves  Figure  6 A shows  a portion  of 
an  infected  leaf  in  the  condition  shown  in  Figure  3 A the  cross 
section  being  taken  at  a point  indicated  by  m.  The  vascular 
bundles  were  all  that  was  left  of  the  leaf  tissues  in  the  infected 
portion  at  this  stage  and  the  fungal  filaments  ramified  through 
all  parts  of  the  bundles  except  the  phloem.  All  of  the  cells  of 
the  mesophyl  had  been  totally  destroyed  and  of  the  epidermal 
cells  the  outer  walls  alone  remained.  A cross  section  through  an 
inner  leaf  of  the  .roll  showed  almost  complete  destruction  of  the 
vascular  bundle  elements  as  is  shown  in  Figure  6 B.  In  all  of 
the  tightly  rolled  and  shriveled  leaf  tips  (See  Figure  3)  the 
dead  and  dried  remnants  of  vascular  bundles  are  closely  bound 
together  by  the  mycelium  which  is  itself  dead  at  this  point. 
During  the  early  part  of  the  season  the  vigorous  destruction  of 
the  host  tissues  proceeds  until  the  plant  often  appears  as  in 
Figure  2,  when  the  entire  roll  of  leaves  is  completely  permeated 
by  mycelium  and  the  destruction  is  so  complete  that  almost  no 
tissues  are  recognized  within  the  roll. 

Action  of  the  Fungus  on  Leaf  Cells  The  action  on  the  in- 
dividual host  cells  is  apparently  rapid.  Careful  study  of  many 
sections  perfectly  fixed,  sectioned  and  stained,  fails  to  show  the 
presence  of  hyphae  within  turgid  mesophyl  cells.  There  is  some 
evidence  that  cells  may  collapse  somewhat  in  advance  of  the  ac- 
tual penetration  of  the  hyphae.  Here  the  plasmolysis  of  cell  con- 
tents preceding  actual  penetration  by  hyphae  is  not  marked  and 
may  be  due  to  other  factors  than  the  direct  influence  of  the  fun- 
gus. .At  any  rate  plasmolysis  and  disintegration  of  the  meso- 
phyl cells  of  the  leaves  occurs  so  rapidly  that  the  successive 
stages  in  the  process  cannot  readily  be  observed. 


224 


Wisconsin  Research  Bulletin  No.  18 


Although,  the  mycelium  develops  abundantly  on  the  leaves  and 
thus  extends  beyond  the  point  of  internal  infection,  there  are 
few  cases  of  penetration  from  without  into  an  expanded  leaf 
either  through  stomata  or  through  the  epidermis.  It  is  notice- 
able that  the  mycelium  spreads  in  the  leaves  most  readily  from 
the  tip  toward  the  base  in  the  direction  in  which  the  vascular 
bundles  run.  This  is  because  the  mycelium  advances  most 
rapidly  in  the  mesophyl  tissues  which  are  arranged  in  strips 
separated  from  each  other  by  the  fibro-vascular  bundles  which 
extend  from  epidermis  to  epidermis  and  across  which  the  myce- 
lium passes  less  readily.  This  is  clearly  shown  in  the  cross  sec- 
tion of  leaves  exhibiting  a strong  lateral  infection.  Such  sec- 
tions reveal  a clearly  defined  boundary  of  the  invasion  which 
presents  for  study  various  stages  in  the  destruction  of  cells.  In 
this  zone  of  advance  through  the  mesophyl  the  mycelium  is 
chiefly  intercellular.  The  ends  and  sides  of  the  hyphae  come  in 
contact  with  the  thin  cell  walls.  In  the  first  stages  of  penetra- 
tion there  appears  to  be  a slight  thickening  of  the  host  cell  wall 
and  a region  about  the  point  of  dissolution  often  stains  strong- 
ly. There  is  no  tendency  for  the  fungus  to  dissolve  out  the 
middle  lamella.  The  hyphae  simpfy  pass  through  openings 
dissolved  in  the  cell  walls.  Once  inside,  their  work  of  destruc- 
tion is  rapid.  The  protoplast  is  plasmolized  and  the  thin  cell 
wall  collapses.  First  the  cell  contents  are  digested  and  later  the 
cell  wall  is  also  completely  digested  so  that  the  entire  mesophyl 
tissue  soon  disappears.  In  the  outer  epidermal  layer  the  my- 
celium often  travels  from  cell  to  cell  destroying  everything  but 
the  layer  of  cuticle. 

In  the  region  of  fungal  advance  the  mycelium  is  less  destruc- 
tive to  the  fibro-vascular  bundles.  Filaments  soon  ramify  freely 
throughout  the  woody  elements  and  there  is  a slight  disorganiza- 
tion of  the  thick  woody  cell  walls. 

In  portions  of  the  leaves  that  have  been  infected  longer  and 
in  the  case  of  inner  leaves  more  vigorously  attacked,  there  is 
almost  complete  destruction  of  the  vascular  elements  as  is  shown 
in  Figure  6 B.  Here  the  woody  walls  are  relatively  thin  and 
collapsed,  and  the  phloem  distorted. 

Distribution  of  the  Mycelium  in  Aerial  Buds  Longitudinal 
sections  were  made  of  terminal  buds  exhibiting  varying  degrees 
of  fungal  destruction.  In  the  early  stages  the  mycelium  ex- 
tends over  the  surface  of  the  rather  tightly  rolled  leaves  with 


A Sclerotium  Disease  of  Common  Grasses 


225 


slight  penetration  in  the  youngest  leaves  and  more  or  less  pen- 
etration in  the  outer.  It  may  even  form  a cushion  of  mycelium 
over  and  around  the  embryonic  tissue  of  the  growing  point  and 
yet  not  penetrate  this  tissue.  Figure  6 D shows  such  a condition. 
Figure  6 E is  a somewhat  diagrammatic  sketch  of  the  same  bud 
from  which  Figure  6 D was  drawn.  It  shows  that  the  myce- 
lium is  both  upon  and  within  the  young  leaves  before  they  un- 
roll from  the  bud.  As  young  leaves  develop  from  the  meriste- 
matic  apex  they  grow  into  this  cushion  of  mycelium  and  are  in 
turn  coated  by  mycelium  and  thus  exposed  to  infection.  As 
noted,  the  mycelium  does  not  penetrate  the  embryonic  tissue. 
In  culms  which  are  vigorously  attacked  the  mycelium  penetrates 
into  the  tissues  of  the  stem  below  the  growing  point,  dissolving 
them  and,  if  the  process  is  not  checked  the  death  of  the  terminal 
bud  may  result. 

The  fungus  seems  first  to  become  destructively  parasitic  in  the 
mesophyl  of  the  outer  leaves  as  they  are  maturing  and  expand- 
ing, to  which  it  gains  entrance  while  the  leaves  are  in  the  bud. 
Then  the  destruction  extends  to  leaves  within  the  bud  roll  and 
finally  to  the  apical  internodes.  In  many  cases,  however,  the 
parasitic  attack  is  confined  to  the  tips  of  the  outer  leaves.  As 
they  develop  each  shows  the  dead  tip  and  the  felt  of  mycelium. 
All  degrees  in  the  rapidity  and  the  amount  of  destruction  may 
be  observed. 

The  Mycelium  in  the  Stems  Plants  were  selected  which 
showed  abundant  fungus  infection  of  the  leaves  and  pieces  were 
cut  from  the  culms  at  various  points.  These  were  sectioned  and 
examined  for  the  presence  of  the  fungus  in  the  successive  modes, 
internodes,  and  buds.  To  the  eye  the  main  portion  of  the  stem, 
with  the  sheathes  of  the  leaves  and  the  buds  enclosed  by  them 
showed  no  felt  of  mycelium  such  as  has  been  described  for  the 
leaves.  The  stained  sections  however,  revealed  the  presence  of 
the  fungus  in  greater  or  less  abundance  in  all  of  these  parts. 
Only  a few  strands  of  hypliae  were  found  in  the  tissues  of  the 
hardened  internodes.  In  the  nodal  regions,  the  strands  were 
quite  numerous  in  the  peripheral  tissues  whe,re  they  often  ap- 
peared as  knotted  tangles  of  intracellular  mycelium.  Occasion- 
ally there  was  penetration  to  the  internodal  cavity.  Here  there 
was  no  evidence  of  destruction  of  cell  walls  other  than  at  the 
points  of  penetration.  In  the  old  cortical  cells  the  cytoplasm  is 


226  Wisconsin  Research  Bulletin  No.  18 

reduced  to  a thin  film  which  is  difficult  to  locate  in  any  of  the 
cells. 

As  a rule  few  buds  are  produced  on  the  upper  parts  of  a 
healthy  culm,  but  late  in  the  season  lateral  buds  often  develop 
from  the  upper  nodes  of  tall  culms  whose  terminal  bud  is  badly 
affected  with  the  fungus.  The  leaves  from  such  buds  may  or 
may  not  be  infected  with  the  fungus. 

The  Mycelium  in  the  Lower  Buds  and  Underground  Stems 
A careful  study  was  made  of  buds  which  arise  low  down  on  the 
culms  and  on  the  rhizomes  and  which  would  not-  develop  into 
culms  or  rhizomes  until  another  season.  It  is  .readily  seen  that 
the  presence  or  absence  of  the  fungus  in  the  case  of  the  buds  is 
a crucial  point  in  determining  the  life  period  of  the  fungus  and 
the  source  of  infection  of  the  unfolding  leaves.  Horizontal  and 
cross  sections  were  made  of  buds  of  different  ages  and  sizes  which 
were  variously  situated  on  basal  portions  of  plants  whose  aerial 
culms  showed  infection.  In  the  majority  of  these  buds  the  myce- 
lium was  found  to  be  present.  Figure  6 G is  a drawing 
from  such  a bud  which  was  situated  just  below  the  surface  of  the 
ground  and  which  would  develop  as  a culm  in  the  following 
season.  The  successive  sections  showed,  as  did  the  longitudinal 
sections  of  other  buds,  that  the  fungus  was  rather  irregularly 
distributed  on  and  through  the  rudimentary  leaves  and  that  it 
was  more  abundant  near  the  apex  of  the  buds.  Often  but  one 
side  of  a bud  was  infected  by  the  fungus.  When  such  a bud  un- 
folds, but  a lateral  half  of  certain  leaves  will  be  diseased,  a con- 
dition j which  explains  the  common  partial  and  lateral  infection 
of  aerial  leaves  already  described  and  illustrated.  In  these  buds 
the  hyphae  pass  through  the  cell  walls  freely  in  the  manner 
shown  in  Figure  6 G.  The  cell  walls  in  all  cases  appear  normal 
but  the  cell  contents  have  entirely  disappeared. 

The  chief  difference  between  the  effects  of  the  fungus  in  the 
foliage  leaves  and  in  the  tissues  of  the  leaves  of  dormant  buds  is 
that  in  the  latter  there  seems  to  be  no  absorption  of  cell  walls 
except  at  the  points  of  penetration. 

In  the  basal  nodes  which  were  close  together  and  from  which 
arose  roots  and  buds  producing  aerial  culms  or  .rhizomes,  the 
fungus  was  abundant.  The  mycelium  was  in  part  external.  Here 
as  in  the  aerial  parts  of  the  stem  itself,  the  fungus  did  not 
penetrate  far  into  the  interior.  In  the  leaf  scales  and  in  the 
cortical  portion  of  the  basal  nodes  there  appeared  within  the 


A Sclerotium  Disease  of  Common  Grasses 


227 


cells,  coils  or  nests  of  mycelium  and  also  rounded  bladder-like 
enlargements  of  the  mycelium,  a feature  not  observed  in  the 
aerial  portions  of  the  host  plant.  These  bladders  often  formed 
a belt  or  zone  in  the  region  of  deepest  penetration,  as  is  shown 
in  Figure  7 A.  The  mycelium  was  traced  readily  from  these 
basal  portions  of  the  stems  out  into  the  buds.  Figures  6 G and 
6 F are  from  a bud  and  the  stem  from  which  it  arose  showing 
the  relative  positions  of  the  two  as  they  appeared  in  the  same 
cross  section.  Figure  7 B is  a section  showing  the  mycelium 
passing  directly  from  the  stem  out  into  a bud  scale  which  en- 
closed a growing  point.  Thus  the  anatomical  studies  verify  the 
experiments  and  field  observations  which  indicated  that  the 
fungus  exists  in  the  underground  buds. 

Relations  of  the  Fungus  to  the  Roots  In  the  stained  sec- 
tions the  fungus  was  also  traced  from  stems  and  rhizomes  out  into 
the  roots,  a fact  which  made  a study  of  the  roots  desirable. 
Plants  of  C.  canadensis  produce  a large  number  of  fine  fibrous 
roots  which  arise  both  from  the  rhizomes  and  from  the  basal 
portions  of  the  culms.  These  form  a dense  tangled  mass  in  the 
surface  layer  of  soil,  especially  in  the  upper  six  inches.  Certain 
roots  may  also  penetrate  to  a depth  of  two  feet.  These  deeply 
penetrating  roots  are  rather  straight,  much  branched,  fibrous 
roots  with  side  branches  which  are  long  and  repeatedly  branch- 
ed. In  contrast  to  these  the  strictly  surface  roots  are  somewhat 
smaller  in  diameter,  shorter,  more  profusely  branched,  and 
much  twisted  and  inte,rwined. 

Many  of  these  roots  live  several  years.  Examination  of  a 
mass  of  roots  in  early  spring  shows  that  some  of  the  roots  are 
dead  while  others  are  putting  out  new  branches.  From  the 
rhizomes  and  from  the  bases  of  living  culms  are  also  produced 
each  spring  new  roots  which  growT  rapidly  to  form  either  the 
deeply  penetrating  roots  or  surface  roots.  Young  and  rapidly 
growing  roots  do  not  harbor  the  mycelium  and  the  deep  roots  do 
not  contain  the  fungus  to  any  considerable  depth.  Many  but 
not  all  of  the  surface  roots  do  contain  the  fungus  and  as  a re- 
sult are  modified  in  a more  or  less  characteristic  manner.  A 
typical  infected  surface  root  that  is  at  least  one  year  old  is 
several  inches  long  with  its  branches  short,  often  twisted  or  curv- 
ed, and  usually  slightly  enlarged.  Root  hairs  are  seldom  found 
on  these  spur  branches,  but  they  are  numerous  along  the  main 


228 


Wisconsin  Research  Bulletin  No.  18 


roots  even  at  points  between  the  side  roots  and  several  inches 
back  of  the  growing  tip.  (See  Figure  8 D.) 

In  making  an  external  examination  of  these  roots  the  mass 
was  soaked  in  water  and  the  soil  was  then  washed  out  in  gently 
running  water.  Then  the  roots  were  mounted  in  water  for  ex- 
amination under  the  low  powers  of  the  microscope.  By  this 
method  the  mycelium  could  be  seen  rather  sparsely  distributed  on 
the  exterior  of  the  roots,  extending  out  from  or  penetrating  in- 
to them,  ramifying  through  the  humus  and  passing  from  root  to 
root.  Few'  filaments  come  directly  from,  the  spur  roots.  Sections 
of  infected  roots  similar  to  the  one  shown  in  Figure  8 D were 
placed  in  hanging  drop  cultures.  In  from  three  to  five  days 
considerable  growth  was  made  by  the  enveloping  myce- 
lium. Many  filaments  were  traced  directly  from  the  roots  and 
often  several  branches  developed  from  the  cut  end  of  a root. 
Figure  8 C represents  the  mycelium  as  it  thus  develops  outside 
a root.  The  drawing  was  made  with  a camera  lucida  using  the 
same  lenses  which  were  used  in  sketching  the  aerial  mycelium 
from  the  leaves  which  is  shown  in  Figure  8 B.  This  mycelium 
appears  to  be  almost  indentical  with  that  produced  on  the  leaves. 

In  examining  roots  by  this  method  mycelium  was  often  found 
infesting  the  roots,  which  was  somewhat  different  in  character 
from  the  foregoing.  It  consisted  in  part  of  large  coarse  hy- 
phae  which  were  sparsely  septate  and  which  had  heavy  walls  of 
unequal  thickness.  On  short  branches  were  borne  terminal 
bladders  or  thick  walled  vesicles  which  were  not  cut  off  by  a 
cell  wall  and  which  we, re  filled  with  oil  globules.  These  vesicles 
we,re  similar  to  those  produced  by  the  mycelium  internally  in 
the  roots,  rhizomes  and  scales  of  the  host  plants,  but  were  much 
larger.  On  casual  examination  this  coarse  mycelium  bears  lit- 
tle resemblance  to  the  mycelium  of  Scleroiium  rhizodes  as  de- 
scribed, but  branches  arising  from  it  may  be  found  which  are 
thin  walled,  smaller  in  diamete.r,  and  more  septate  and  almost 
identical  with  those  which  were  secured  from  the  cut  roots  in 
the  hanging  drop  cultures.  This  heavy  walled  mycelium  with 
the  numerous  large  food  vesicles  was  found  on  roots  of  infected 
plants  collected  from  widely  separated  localities  at  various  times 
throughout  the  spring,  summer  and  autumn. 

The  evidence  is  conclusive  that  the  fungus  is  in  pa,rt  soil  in- 
habiting and  this  explains  the  vigorous  local  infection  as  noted 
and  the  infection  of  a number  of  grasses  which  may  enter  such 


A Sclerotium  Disease  of  Common  Grasses 


229 


an  area,  especially  the  infection  of  an  annual,  such  as  Hordeum 
jubatum.  A large  number  of  roots  of  various  sizes  and  ages 
were  collected  from  infested  C.  canadensis  plants  at  intervals 
throughout  the  season  of  1910,  fixed  in  picro-formal  solution, 
imbedded  in  paraffin,  sectioned  with  a microtome,  and  stained  by 
the  triple  method.  These  sections  revealed  the  presence  of  the 
fungus  within  nearly  all  of  the  older  surface  roots.  Young 
rapidly  growing  roots  did  not  contain  the  fungus. 

So  far  as  the  behavior  here  is  concerned  there  is  no  evidence 
that  there  is  present  a mycorrhizal  relationship  such  as  is  com- 
monly understood  in  which  the  fungus  retains  a constant  posi- 
tion in  reference  to  the  growing  points.  In  the  roots,  rhizomes, 
and  stems  the  fungus  attacks  the  older  and  hence  weakened  cells. 
This  is  in  marked  contrast  to  the  action  in  the  leaves  where  it 
attacks  vigorously  the  active  mesophyl  cells  but  it  is  to  be  noted 
that  the  hyphae  are  unable  to  penetrate  into  the  growing  apex 
of  the  buds. 

In  larger  roots  such  as  shown  in  Figure  8 D there  was  an 
abundant  but  rather  irregular  distribution  of  the  mycelium 
throughout  the  cortical  tissue,  with  much  the  same  characteris- 
tics as  are  seen  in  the  cortex  of  the  underground  stems.  The 
nests  of  mycelium  and  the  bladders  were  formed  within  the  cells 
although  the  latter  were  not  grouped  in  a belt  or  zone.  The  my- 
celium was  both  inter-and  intra-cellular.  In  the  root  cells  of  this 
region  the  cytoplasm  forms  an  extremely  thin  layer  which  was 
difficult  to  identify  even  in  uninfected  cells.  The  cell  walls 
were  of  normal  thickness  and  were  not  collapsed.  There  was  a 
strong  tendency  for  the  mycelium  to  extend  longitudinally 
through  the  root  yet  there  was  here  some  evidence  of  its  spread 
in  a radial  direction  also.  (See  Figure  ID.) 

In  the  spur  roots  there  is  considerable  variability  in  the  be- 
havior of  the  fungus.  Here  the  mycelium  is  confined  chiefly  to 
the  layer  of  cells  immediately  surrounding  the  central  cylinder. 
It  can  be  traced  into  this  zone  from  the  main  root  and  its  dis- 
tribution is  here  decidedly  in  the  direction  of  the  growth  of  the 
rootlet.  Here  there  is  an  opportunity  to  study  the  relations 
of  the  hyphae  to  the  protoplast  for  they  penetrate  cells  while 
the  cytoplasm  is  conspicuous  and  the  cell  walls  are  thin.  The 
fungus  advances  to  the  extreme  tip  which  is  devoid  of  a pro- 
nounced embryonic  region.  When  a liypha  passes  into  a living 
cell  in  this  region  it  first  becomes  irregularly  enlarged  or  lobed 


280 


Wisconsin  Kesearch  Bulletin  No.  18 


and  is  somewhat  coiled  about  or  pressed  upon  the  nucleus  which 
m turn  becomes  rather  irregular  in  shape.  The  cytoplasm  be- 
comes dense  and  granular  or  slightly  stringy  and  stains  a deep 
orange  or  red.  (Figure  7 C,  E and  G.)  Late,r  the  nucleus 
disappears  in  some  cases  evidently  after  fragmenting  and  the 
cytoplasm  is  transformed  into  irregular,  dense,  deeply  staining 
particles  scattered  about  within  the  cell  wall.  (See  7 F).  In 
some  instances  the  fungus  filaments  within  such  a cell  seem  also 
to  disintegrate  so  that  fragments  of  the  mycelium  are  mingled 
with  the  debris  of  the  protoplast.  In  the  majority  of  infected 
cells,  however,  the  mycelium  remains  intact  while  the  cytoplasm 
and  nucleus  are  undergoing  disintegration.  Adjoining  but  un- 
infected cells  show  the  cytoplasm  and  nucleus  as  normal  and 
faintly  staining  structures. 


In  certain  of  these  infected  cells  these  changes  in  the  pro- 
toplast are  accompanied  by  the  formation  of  bladders  which  are 
intercalary  or  perhaps  occasionally  terminal  swellings  of  the 
hyphae.  They  are  always  intracellular  and  are  of  various  sizes. 
They  begin  to  appear  in  the  small  cells  of  roots  in  the  region 
of  the  advance  of  the  fungus  and  they  may  be  found  fully  de- 
veloped in  the  older  roots  and  in  the  basal  nodes  and  the  sur- 
rounding scales.  When  young  they  show  a finely  reticulated 
structure  (Figure  8 F .)  Later  they  are  more  dense  and  gran- 
ular. The  greater  number  found  in  old  roots  are  entirely  empty 
and  possess  rigid  and  unbroken  cell  walls  although  in  a few 
cases  they  appear  wrinkled  or  shriveled. 


In  early  spring  the  vigorously  growing  roots  are  not  infected. 
Soon  these  roots  cease  their  rapid  growth  and  begin  to  send 
out  short  slowly  growing  side  roots.  The  fungus  mycelium 
probably  gains  entrance  both  at  the  base  from  the  stem  and  by 
direct  penetration  from  the  soil.  It  advances  in  the  root  toward 
the  growing  point.  The  lateral  roots  are  infected  directly  from 
the  main  root  soon  after  they  are  first  formed  and  as  a result 
are  stunted  and  slightly  hypertrophied.  These  infected  roots 
may  continue  to  live  into  at  least  the  second  season.  Some  side 
roots  escape  infection  completely  or  for  a longer  time  and  these 
elongate  in  a normal  fashion. 

^ Summary  of  the  Relations  of  the  Host  and  the  Fungus 
These  studies  show  that  the  fungus  is  coexistent  in  leaves,  stems, 
buds,  rhizomes  and  roots  of  the  same  plant.  Its  general  distribu- 
tion m underground  perennial  parts  and  its  existence  in  buds 


A Sclerotium  Disease  of  Common  Grasses  231 

which  are  to  develop  in  succeeding  seasons  make  clear  the  peren- 
nial nature  of  the  mycelium.  It  is  of  especial  significance  that 
the  mycelium  was  found  in  greater  or  less  abundance  in  the 
scales  and  young  leaves  immediately  surrounding  growing 
points.  This  provides  for  the  distribution  of  the  mycelium 
into  the  various  branches  as  they  are  formed.  As  an  infected 
basal  bud  develops  into  a culm  the  intercalary  growth  incident 
to  the  development  of  the  internodes  separates  the  leaves  which 
are  more  or  less  infected  in  the  bud.  As  the  internodes  elong- 
ate, the  mycelium,  which  as  has  been  noted,  is  chiefly  confined 
to  the  young  leaves,  is  carried  with  them  and  hence  appears  on 
the  successive  leaves.  The  tips  however,  being  held  together  by 
the  fungus  form  the  series  of  characteristic  crooks. 

This  development,  with  the  quite  general  distribution  of  the 
mycelium  in  the  soil  and  in  the  underground  portions  of  the 
host,  insures  most  completely  its  perennial  habit  and  accounts 
for  its  reappearance  without  spore  formation  in  the  same  areas 
year  after  year,  for  the  decided  local  infection  so  often  ob- 
served, and  for  the  infection  of  various  other  grasses  which  enter 
the  infected  areas.  I have  not  yet  worked  out  the  relations  of 
the  fungus  to  the  leaves,  roots,  and  stems  of  the  various  other 
grasses  upon  which  it  has  been  found,  so  fully  as  in  the  case  of 
C.  canadensis  but  the  evidence  obtained  indicates  that  the  gen- 
eral relationships  of  host  and  fungus  are  the  same  in  each  in- 
stance. 

There  is  in  the  case  of  the  leaves  of  all  infected  species  the 
same  characteristic  development  of  mycelium,  production  of 
sclerotia,  formation  of  crooks  and  death  to  the  leaves.  The 
leaves  of  Poa  pratensis,  however,  show  a more  decidedly  local 
infection.  Often  two  or  more  infected  spots  will  be  completely 
separated  by  green  tissue.  This  is  evidently  due  to  an  irregu- 
lar infection  in  the  bud  and  to  the  inability  of  the  fungus  to 
spread  so  rapidly  in  the  leaves  of  this  species  as  it  does  in  other 
grasses. 

In  the  roots  arising  from  infected  culms  of  C.  neglecta  there 
is  a general  distribution  of  the  fungus  with  the  same  character- 
istics and  effects  described  for  C.  canadensis. 

Sections  w^ere  also  made  through  clusters  of  roots  on  infected 
culms  of  Poa  pratensis,  Panicularia  n'ervata  and  Hordeum  juba- 
tum..  In  all  of  these  the  fungus  was  found  in  and  on  roots, 


232 


Wisconsin  Research  Bulletin  No.  18 


scales,  and  underground  portions  of  the  stem,  although  not  to 
such  an  extent  as  was  found  in  C.  canadensis. 

Sclerotia 

The  development  of  sclerotia  was  observed  on  the  host  plant 
and  on  various  culture  media.  They  originate  in  a small  loose 
plexus  of  several  mycelial  strands  which  continue  ,to  branch, 
intertwine  and  anastomose  until  a .rather  compact  mass  of 
tissue  is  formed.  The  cells  within  become  enlarged  and 
irregularly  rounded.  The  young  sclerotium  increases  in  size  by 
repeated  growth  and  branching  from  the  periphery.  Meanwhile 
large  drops  of  clear- liquid  of  a slightly  yellow  color  are  exuded. 
In  maturing  the  color  changes  from  white  to  dark  brown  o,r  black 
and  the  outermost  zone  of  filaments  shrivels  and  forms  a thin 
felted  layer  beneath  which  a dark  colored  rind  develops.  In 
this  rind  the  cells  are  small  and  nearly  isodiametic  and  possess 
heavy  walls.  The  central  mass  is  made  up  of  intertwined  hy- 
phae  whose  cells  a, re  shortened;  and  of  greater  diameter  than 
those  of  the  ordinary  aerial  mycelium.  Although  rather  closely 
packed  together  there  are  many  spaces  between  them.  The 
structure  appears  homogeneous  with  no  trace  of  primordia. 

Two  o.r  more  young  sclerotia  which  are  growing  close  together 
often  unite  to  make  an  irregularly  lobed  compound  sclerotium. 
In  cultures  on  cooked  potato  and  on  potato  agar  the  small  scle 
rotia  were  so  numerous  that  they  formed  a crusted  stroma  which 
did  not  round  out  into  any  definite  shape.  On  the  various  other 
media  tested  there  developed  every  stage  from  a matted  myce- 
lium forming  an  indefinite  stroma  to  well  rounded  sclerotia.  In 
form,  many  of  these  irregularly  shaped  sclerotia  resemble  those 
which  Brefeld  (1881  p.  115)  obtained  in  his  cultures  of  Peziza 
sclerotioriim.  Brefeld  (1881  p.  116)  has  also  most  clearly 
pointed  out  the  two  methods  of  origin  of  sclerotia.  The  sclerotia 
of  Basidiomycetes  (Agaricus,  Coprinus,  Typhula,  etc.)  start  by 
the  interlacing  of  branches  from  a single  hypha,  while  the  scle- 
rotia of  Ascomycetes  which  he  investigated  begin  as  a plexus  of 
several  filaments,  as  previously  noted  for  this  fungus. 

The  sclerotia  which  develop  on  the  host  plants  are  rounded 
and  smooth  on  thei,r  entire  surface  except  on  the  side  which  was 
appressed  to  the  leaf  and  here  the  sclerotium  is  usually  flattened 
and  rugose  to  conform  to  the  ridges  in  the  surface  of  the  leaf. 


A Sclerotium  Disease  of  Common  Grasses 


233 


When  fully  mature  they  usually  drop  from  the  leaf.  Attached 
to  many  of  them  are  often  short  strings  of  dry  and  dead  myce- 
lium from  which  the  sclerotium  arose  and  in  which  the  growing 
sclerotium  is  imbedded.  These  appear  on  the  immature  sclero- 
tia  especially  somewhat  like  roots,  or  rhizoids,  and  hence  prob- 
ably suggested  the  specific  name  given  by  Auerswald. 

It  seems  probable  that  spores  when  they  are  produced  develop 
first  saprophytically  in  the  humus  soil.  Investigations  and  ex- 
periments are  still  in  progress  to  determine  more  completely  the 
ultimate  fate  of  the  sclerotia.  As  many  as  500  sclerotia  of  the 
1911  crop  are  now  planted  in  pots  and  in  bottles  which  are  be- 
ing handled  in  a variety  of  ways. 

There  is  some  evidence  that  sclerotia  may  sprout  vegetatively. 
Fully  mature  sclerotia  were  taken  before  they  had  dried,  and 
placed  on  sterile  sand  in  small  pots  which  were  covered  with 
glass  lids  and  kept  moist.  A thin  felt  of  mycelium  developed 
from  the  sclerotia  and  spread  over  the  sand  where  it  continued 
to  thrive  for  several  months.  When  bits  of  this  mycelium  were 
transferred  to  various  media  the  typical  growth  resulted.  Other 
sclerotia  were  placed  directly  on  media  and  in  the  majority  of 
cases  developed  a growth  typical  to  the  various  media.  Old 
sclerotia  which  were  thoroughly  dried  failed  to  produce  growth 
of  any  kind  when  thus  treated. 

Infection  Experiments 

For  these  experiments  healthy  uninfected  plants  of  C.  cana- 
densis were  grown  from  rhizomes  transplanted  to  pots  of  sand  or 
garden  loam.  When  the  culms  were  from  three  to  twelve  inches 
m height  masses  of  mycelium  from  various  cultures  were  placed 
on  leaves  of  various  ages  and  the  plants  were  then  kept  in  bell 
jars.  In  no  case  did  the  mycelium  establish  itself  on  the  leaves. 
The  following  method  was  then  tried.  Vigorously  growing  pure 
cultures  were  grown  on  hard  potato  agar  and  on  lima  bean  agar 
in  test  tube  slants.  A test  tube  with  a culture  was  inverted,  the 
cotton  plug  removed  and  the  open  tube  slipped  over  a culm  and 
so  adjusted  in  a clamp  stand  that  the  leaves  came  in  contact  with 
the  mycelium.  The  plug  was  then  replaced  and  thus  the  unin- 
jured mycelium  was  brought  in  contact  with  leaves  and  the 
whole  was  enclosed  in  a moist  chamber  formed  by  the  plugged 
test  tube.  In  all,  twenty-five  experiments  were  tried  on  culms 


234 


Wisconsin  Research  Bulletin  No.  IB 


of  various  ages  and  on  leaves  in  various  stages  of  development. 
The  mycelium  would  develop  over  and  about  the  leaves  and  al- 
though left  for  several  days,  when  the  test  tube  with  its  culture 
was  removed  and  the  culm  enclosed  in  a moist  chamber,  the 
mycelium  adhering  to  the  leaves  died,  the  leaves  continued  to 
develop  normally  and  there  was  no  evidence  of  infection. 

Infected  culms  were  brought  in  from  the  field  and  enclosed  in 
a bell  jar  until  there  was  abundant  development  of  aerial  myce- 
lium and  then  placed  in  contact  with  healthy  leaves.  No  infec- 
tion resulted  by  this  method.  Experiments  to  test  the  possibil- 
ity of  the  infection  of  seedlings  through  the  soil  are  now  being 
carried  on  and  have  not  yet  given  any  definite  results.  These 
experiments  seem  to  indicate  clearly  that  the  mycelium  can  not 
penetrate  into  sound  leaves  after  they  have  opened  from  the  bud 
and  that  there  is  no  spread  of  the  fungus  from  culm  to  culm 
aerially. 

Cultural  Studies 

The  fungus  was  obtained  in  pure  culture  by  placing  fragments 
of  infected  leaves  in  Petri  dishes  poured  with  lima  bean  agar 
and  with  hard  potato  agar.  Abundant  growth  of  mycelium  with 
formation  of  sclerotia  resulted  and  transfers  were  readily  made 
to  media  in  test  tubes.  The  fungus  has  been  kept  in  culture 
since  April  1910  and  its  behavior  on  various  media  studied.  All 
cultures  were  grown  in  a refrigerator  at  a temperature  of  about 
16°  C. 

Lima  Bean  Agarz  On  this  medium  there  is  luxuriant  and 
rapid  growth.  At  first  a fine  cottony  layer  of  mycelium  spreads 
over  the  surface  of  the  medium  and  from  this  there  is  a copious 
aerial  growth.  Within  from  ten  to  twenty  days  sclerotia  begin 
to  form.  When  mature  many  of  these  are  rounded  and  similar 
in  size  to  those  formed  on  the  host  in  the  field,  but  the  individual 
sclerotia  may  grow  together  to  make  an  irregular  mass,  often 
1.5  cm.  in  diameter.  Several  cultures  were  kept  for  a period  of 
15  months  and  although  the  medium  had  shrunken  to  one-third 
of  its  original  volume  the  mycelium  was  still  vigorous  in  small 
patches  and  new  sclerotia  were  being  formed. 


s Lima  bean  agar:  1000  cc.  water,  100  g.  ground  beans;  soak  30  min., 
then  boil  30  min.;  express  juice  and  restore  to  1000  cc.;  add  10  g.  agar, 
cook  in  steamer,  filter,  autoclave  at  5 lbs. 


A Sclerotium  Disease  of  Common  Grasses 


235 


Hard  Potato  Agar 4 Growth  was  less  rapid  on  this  medium 

that  that  obtained  on  lima  bean  agar.  A few  definite  sclerotia 
were  formed  but  as  a rule  large  ir, regular  spongy  masses  formed 
on  the  surface  of  the  medium  as  a crusted  stroma.  The  surface 
of  these  remained  spongy  or  granular  ; from  the  surface  there 
constantly  developed  tufts  of  white  mycelium  which  in  turn 
became  brown,  rough  and  granular.  The  largest  of  these 
pseudo-sclerotia  were  3 cm.  long  and  .5  cm  thick.  They  con- 
sisted of  a loose  plexus  of  mycelium  the  cells  of  which  were  en- 
laged  like  those  of  young  sclerotia.  (Figures  4 A and  5 A). 
Granular  portions  of  these  pseudo-sclerotia  when  broken  out 
and  transferred  to  various  media  always  gave  the  characteris- 
tic development. 

Cooked  Bean  Pods  The,re  was  abundant  development  on 
cooked  bean  pods  with  formation  of  many  rounded  perfect 
sclerotia  which  were  as  a rule  larger  than  the  majority  of  those 
formed  on  C.  canadensis  in  the  field.  The  sclerotia  were  always 
external  to  the  pod.  Sections  of  the  bean  pods  thus  infected 
were  made  and  it  was  found  that  the  mycelium  penetrated  the 
cell  walls  and  coiled  about  in  the  cells.  The  visible  effect  on  the 
pod  was  simply  that  of  shriveling.  The  mycelium  grew  vigor- 
ously for  several  months,  penetrated  to  every  part  of  the  pod 
and  seeds  and  there  was  also  considerable  aerial  development. 

On  sterilized  culms  and  leaves  of  C.  canadensis  and  Dactylis 
glomerata  the  fungus  grew  vigorously  and  many  rounded  ma- 
ture sclerotia  we, re  produced. 

Bouillon  There  was  a slow  but  considerable  aerial  growth 
on  bouillon  which  spread  over  the  surface  of  the  medium.  No 
sclerotia  were  formed. 

Nutrient  Gelatin  An  abundant  growth  wms  obtained  on 
this  medium,  but  only  a few  small  sclerotia  were  developed. 

Cooked  Potato  The  growth  spread  over  the  surface  of 
cooked  potato  as  a thin  but  rather  compact  granular  layer  which 
was  similar  to  the  pseudo-sclerotia  produced  on  the  hard  potato 
agar. 

Ashby’s  Medium  When  this  was  poured  on  pure  quartz 
sand  and  inoculated  there  was  a steady  growth  of  mycelium 

4 Hard  potato  agar:  1000  cc.  water,  200  grams  of  sliced  potato  tuber; 
cook  in  steamer  1 hr.;  strain  off  clear  liquid  and  restore  to  1000  cc.; 
add  20  g.  glucose  and  30  grams  agar;  cook  in  steamer  1 hr.,  filter, 
autoclave. 


236 


Wisconsin  Research  Bulletin  No.  18 


which  spread  ove,r  the  surface  with  considerable  aerial  develop- 
ment. The  development  of  sclerotia  was,  however,  feeble. 

Diast atic  Action  To  test  the  action  of  the  fungus  on  starch 
the  following  method  was  used.  To  each  of  three  tubes  of  hard 
potato  agar  and  three  tubes  of  lima  bean  agar,  there  was  added 
one  gram  of  finely  powdered  potato  starch.  The  tubes  were 
placed  in  an  autoclave  until  the  medium  was  liquid  when  the 
starch  was  thoroughly  mixed  by  shaking  and  then  poured  into 
Petri  dishes.  A transfer  of  mycelium  was  made  to  each  of  the 
plates  and  an  unusually  vigorous  growth  of  mycelium  resulted. 
When  this  growth  covered  an  area  of  about  4 cm.  in  diameter 
iodine  in  potassium  iodide  solution  was  poured  into  four  of  the 
plates,  two  of  each  medium,  and  allowed  to  stand  several  min- 
utes. When  this  was  rinsed  the  portion  covered  with  the  mycel- 
ium in  all  fou,r  plates  showed  white  with  only  an  occasional 
granule  of  blue,  while  the  entire  surface  of  the  medium  outside 
of  the  border  of  the  fungus  was  a deep  purple.  Thus  it  is 
clear  that  the  fungus  can  digest  cooked  starch  in  culture  media. 
The  other  two  plates  were  allowed  to  develop  at  length  and  on 
these,  the  fungus  made  an  unusually  vigorous  growth.  It  rapidly 
spread  over  the  entire  surface  of  the  medium  and  up  the  sides 
of  the  dish  to  the  cover.  Many  small  sized  sclerotia  and  several 
unusually  large  ones  were  formed.  These  developments  showed 
that  the  addition  of  starch  to  culture  media  increases  the  vigor 
of  growth  and  the  formation  of  sclerotia. 

Pure  Cultures  in  Soil  The  fungus  was  grown  on  ordinary 
marsh  soil  by  the  following  method : masses  of  the  top  layers 
of  peaty  soil  upon  which  infected  plants  of  C.  canadensis  had 
been  growing  we,re  placed  intact,  (with  various  grass  roots,  etc., 
included  as  in  the  soil)  in  test  tubes  and  bottles  which  were 
plugged  and  sterilized  in  an  autoclave  at  15  pounds  for  twenty 
minutes.  Transfers  of  mycelium  from  pure  cultures  to  this  soil 
resulted  in  a steady  healthy  growth  of  mycelium  which  pene- 
trated the  loose  soil  in  all  directions  (See  Figure  5 B).  Many 
rounded  sclerotia  some  of  which  were  5 m m.  in  diameter  devel- 
oped both  at  the  surface  and  at  various  depths  in  the  tubes  of 
soil.  Soil  cultures  of  this  description  are  now  being  used  for 
experiments  to  test  the  infection  of  young  seedlings. 


A Sclerotium  Disease  of  Common  Grasses 


237 


Characteristics  of  the  Fungus  ; Discussion  and  Comparisons 

The  fungus  Sclerotium  rhizodes  in  its  relations  to  the  peren- 
nial grasses  studied  exhibits  a combination  of  characteristics  not 
hitherto  ascribed  to  any  one  fungus. 

In  the  aerial  parts  of  the  host  the  behavior  is  somewhat  simi- 
lar to  that  of  various  smuts,  especially  as  shown  by  the  recent 
studies  of  Lutman  (1910,  p.  1204).  He  found  for  Ustilago  levis 
on  oats  that  in  the  growing  plants  the  mycelium  is  most  abund- 
ant at  the  nodes  and  in  the  growing  points  and  that  many  leaves 
contained  the  fungus  to  their  tips.  The  mycelium  remains  in 
or  near  the  growing  points  but  is  intercellular. 

McAlpine  (1910,  p.  10)  has  pointed  out  the  tendency  for 
the  mycelium  of  Ustilago  nuda  on  barley  to  persist  in  the  under- 
ground portions  of  the  plants  and  penetrate  into  new  culms 
which  were  induced  by  repeated  cutting  of  the  old  culms. 

The  seed  fungus  of  Lolium  temulentum  as  described  by  Free- 
man (1903,  p.  14-16)  shows  quite  a different  behavior.  Here  the 
fungus  seldom  enters  the  roots  and  leaves,  but  lingers  in  the 
growing  points  and  finally  enters  the  nucellus  layer  of  the  seed 
from  which  it  later  infects  the  germinating  embryo.  There  is, 
however,  no  destructive  effect  on  the  seed;  in  fact,  Freeman 
notes  (page  5)  that  infected  seeds  are  larger  and  better  devel- 
oped, suggesting  a symbiotic  relationship  which  Hiltner’s  (1899, 
p.  835-837)  work  seems  to  show  is  due  to  the  fixation  of  atmos- 
pheric nitrogen  by  the  fungus. 

Miss  Ternetz  (1907,  p.  359)  in  her  studies  of  various  mycor- 
rhiza  finds  the  seeds  of  the  macrosymbionts  infected  and  raises 
the  question  as  to  the  source  of  this  infection.  In  Andromeda 
polifolia  she  observes  mycelium  in  the  rind  of  the  older  twigs 
which  had  reached  this  point  by  the  coordinated  growth,  of  the 
plant  and  the  mycelium.  Thus  she  finds  the  mycelium  wander- 
ing from  roots  into  aerial  parts,  but  she  notes  that  it  avoids 
chlorophyl  bearing  cells.  Since  she  did  not  find  mycelium  in 
the  flower  stalks  she  believes  that  the  source  of  the  infection 
in  the  flowers  is  external,  yet  it  seems  probable  from  the  positive 
evidence  given  above,  that  this  infection  of  the  fruit  comes 
vegetatively  from  the  roots. 

As  to  its  distribution  within  the  host,  Phoma  radicis  An* 
diomedae,  as  described  by  Ternetz  seems  to  be  intermediate  be- 


238 


Wisconsin  Research  Bulletin  No.  18 


tween  strictly  root  inhabiting  mycorrhiza  and  Sclerotium  rhiz- 
odes  on  Calamagrostis  canadensis  as  described. 

Turning  to  the  behavior  of  Sclerotium  rhizodes  in  roots  and 
underground  stems,  we  find  that  its  morphological  characters 
and  host  relations  a, re  such  that  it  might  be  considered  as  a 
myeorrhizal  fungus.  This  term,  however,  has  been  so  loosely 
applied  to  root  inhabiting  fungi  that  it  has  at  present  little 
specific  significance.  Following  the  rather  limited  observations 
of  Kamienski  (1881  and  1882),  Frank  (1885  and  1887),  and 
Woronin  (1885),  the  more  extensive  studies  especially  by 
Schlicht  (1889),  Janse  (1896),  and  Stahl  (1900)  have  shown 
that  root  inhabiting  fungi  are  present  in  a large  numbe,r  of 
flowering  plants.  So  called  mycorrhiza  have  also  been  found 
in  the  tissues  of  certain  cryptogams  as  has  been  shown  especially 
by  Atkinson  (1893),  Janse  (1896),  Nemec  (1899),  Lang  (1899) 
and  Campbell  (1908). 

It  has  been  established  that  annuals,  biennials,  and  perennials 
growing  in  all  sorts  of  soil,  belonging  to  a wide  range  of  fami- 
lies and  exhibiting  autotropic  as  well  as  holosaprophytic  modes 
of  life,  may  possess  root  inhabiting  fungi  of  endotropic  or  ec- 
totropic  character. 

Schlicht  (1899,  p.  26)  includes  in  his  list  of  species  possess- 
ing endotropic  mycorrhiza  the  grasses  IIolcus  lanatus,  Festuca 
ovina,  Agrostis  caninam , Aira  caespitosa  and  Triodia  dccum- 
bens.  To  these  Stahl  (1900,  p.  551,  552)  adds  the  following 
grasses:  Sesleria  coendea,  Agrostis  alpina,  Brachy podium  pin- 
natum,  Molinea  coendea.  It  appears  that  with  the  exception  of 
IIolcus  lanatus  none  of  these  grasses  have  been  investigated  as 
to  the  character  and  behavior  of  their  myeorrhizal  fungi.  In 
fact  but  few  of  the  large  list  of  species  possessing  so-called 
mycorrhiza  have  been  thus  studied  and  it  is  by  no  means  clear 
that  there  is  a sharp  distinction  between  symbiotic  mycorrhiza 
and  the  parasitic  and  saprophytic  forms. 

Schlicht  (1899,  pp.  17-18)  has  described  somewhat  in  detail 
the  root  fungus  which  he  found  in  the  grass  IIolcus  lanatus. 
Here  the  fungus  coated  the  exterior  of  the  finer  roots  with  a 
thin  sparsely  distributed  felt  of  dark  brown  mycelium  from 
which  fibers  penetrated  into  the  inner  cortex  where  they  formed 
a sort  of  mantle  about  the  central  cylinder.  lie  found  knots 
of  filaments,  swollen  portions  of  the  mycelium,  and  in  the  older 
parts  of  the  root  irregular  masses  of  disintegrating  fungus.  He 


A Sclerotium  Disease  of  Common  Grasses  239 

noted  that  the  cell  nucleus  can  exist  in  the  same  cells  in  which 
there  is  a plexus  of  filaments.  Thus  fa,r  his  description  and 
drawing  (1899,  Figure  12)  shows  almost  identically  the  same 
conditions  found  by  the  writer  in  the  roots  of  C.  canadensis . 
The  manner  of  penetration  and  the  general  character  of  the  fun- 
gus seem  identical  in  both  cases.  Schlicht,  however,  considered 
that  the  fungus  in  Holcus  lanatus  was  limited  almost  entirely 
to  the  small  roots.  Although  he  noted  the  presence  of  the  fun- 
gus in  the  older  parts  of  the  main  roots  he  considered  that 
here  the  mycelium  went  into  a resting  condition  or  died,  or  at 
any  rate  lost  its  mycorrhizal  character.  He  found  no  evidence 
of  parasitic  behavior  in  the  roots  or  of  abnormal  conditions 
and  he  argues  at  length  for  a symbiotic  relationship.  He  did  not 
discover  any  mycelium  in  the  rhizome,  bud  or  leaf  as  it  exists  in 
C.  canadensis. 

Schlicht  collected  his  material  about  the  year  1888  in  the  vic- 
inity of  Berlin,  a region  in  which  Sclerotium  rhizodes  had  al- 
ready been  found  on  a number  of  grasses.  It  is  also  to  be  noted 
that  in  1891  the  fungus  Sclerotium  rhizodes  was  collected  (Krie* 
ger,  Fungi  Saxonici  No.  1399)  on  Holcus  lanatus  within  100 
miles  of  Berlin.  In  view  of  the  results  here  reported,  it  seems 
probable  that  the  root  fungus  as  described  by  Schlicht  is  Scleio - 
tium  rhizodes.  The  presence  of  the  fungus  on  the  leaves  as  it  ap- 
pears in  Wisconsin  on  the  various  grasses  might  easily  be  over- 
looked, especially  late  in  summer,  unless  there  was  a severe 
epidemic.  Even  when  noticed  on  the  leaves  one  would  not  be 
likely  to  associate  it  with  a root  inhabiting  fungus. 

Groom  (1895)  working  on  Thismia  Aseroe,  which  is  a liolo- 
saprophyte,  finds  a still  more  marked  differentiation  of  the  fun- 
gus in  the  various  tissues.  He  does  not  observe  any  destructive 
effects  due  to  the  fungus.  Conspicuous  bladders  and  coils  of 
mycelium  are  formed  at  the  expense  of  certain  host  cells,  but 
these  he  considers  break  down  when  mature  thus  returning  to 
the  cells  certain  food  materials  after  which  the  cells  resume  their 
normal  activity.  According  to  this  the  vesicles  here  formed  d> 
not  furnish  food  to  the  mycelium  outside  of  the  particular  cells 
in  which  they  are  situated. 

Magnus  (1900)  described  the  conditions  which  exist  in  the 
non-chlorophyl  bearing  plant  Neottia  nidus  avis.  In  the  cortex 
of  the  roots  and  the  rhizome  he  finds  an  endotropic  mycorrhizal 
fungus.  In  certain  cells  the  fungus  destroys  the  protoplast  and 


240  Wisconsin  Research  Bulletin  No.  18 

forms  thick  walled  hyphae  and  bladder-like  structures  which 
he  considers  to  be  organs  for  storage  of  food.  In  the  outer  and 
inner  layers  of  the  zone  of  infection,  however,  he  finds  digesting 
cells  in  which  the  fungus  is  for  the  most  part  digested.  Thus 
he  finds  that  the  fungus  is  locally  parasitic  in  some  cells  and 
that  in  others  it  yields  its  substance  to  the  host  cells  which  con- 
tinue to  live. 

Shibata  (1902)  finds  much  the  same  condition  in  Podocarpus, 
Alnus,  and  Psilotum.  In  the  digesting  cells  of  the  last  two 
named  the  wall  substance  of  the  fungus  remains  as  densely  stain- 
ing clumps.  In  the  cells  destroyed  by  the  fungus  he  notes  the 
formation  of  vesicles  which  are  filled  with  food. 

Arzberger  (1910)  has  recently  investigated  the  root  tuber- 
cles of  Ceamcihus  americmus,  Elaeagnus  argentea,  and  Myrica 
cerifera.  He  finds  in  Ceanothus  that  hypertrophied  cells  and 
nuclei  result  from  the  infection,  that  the  cell  walls  of  host 
cells  are  dissolved  and  that  later  the  cytoplasm  and  nuclei  of 
these  cells  are  absorbed.  Soon  the  cell  content  of  the  fungus  dis- 
integrates, but  not  until  the  contents  of  the  host  cell  are  used  up. 
In  the  root  tubercles  of  the  above  named  species  he  finds  ulti- 
mately both  the  host  cell  and  fungus  die  “as  a result  of  their 
previous  relationship,”  but  he  concludes  that  the  material  is  in 
some  way  used  by  the  adjoining  healthy  uninfected  cells. 

Regarding  the  vesicles  or  “sporanges”  which  are  formed, 
Arzberger  finds  them  to  be  terminal,  spherical  or  pear  shaped 
bodies.  In  Ceanothus  and  Myrica  their  contents  break  up  into 
a few  segments  by  a process  which  he  considers  analogous  to 
spore  formation  in  other  fungi. 


Physiological  Significance 

In  all  of  the  cases  described  by  these  authors,  with  the  excep- 
tion of  Schlicht,  in  some  cells  at  least  the  fungus  is  wholly  or 
in  part  destructive.  They  all  find  evidence,  however,  of  a sym- 
biotic relationship  in  the  digesting  cells  or  in  the  benefit  which 
the  surrounding  cells  may  gain.  The  anatomical  evidence  for 
fsymbiosis  in  the  case  of  endotropic  mycorrhiza  seems  to  rest  on 
the  observed  ability  of  individual  cells  to  recover  from  the  in- 
vasion of  the  fungus  or  in  the  ability  of  adjoining  cells  to  pro- 
fit by  the  activities  of  the  fungus  though  it  may  be  destructive 
of  certain  individual  cells. 


A Sclerotium  Disease  of  Common  Grasses  241 

The  characteristics  of  these  endotropic  fungi  within  the  cells 
during  the  early  stages  of  infection  and  the  formation  of  blad- 
ders as  described  by  Magnus  (1900,  pp.  10-19),  Shibata  (1902, 
pp.  646,  657-660)  and  most  especially  by  Groom  (1895  pp.  333, 
334  and  338-340)  bear  some  noticeable  resemblance  to  Sclero- 
tium rhizodes  although  it.  is  to  be  noted  that  the  macrosymbionts 
referred  to  above  range  from  autotropic  to  holosaprophytic 
plants. 

The  later  developments  of  the  Sclerotium  rhizodes  in  the  roots 
suggest  a somewhat  different  relationship.  I find  no  conclusive 
evidence  that  there  are  here  digesting  cells  which  overcome 
the  fungus  and  resume  a normal  appearance  after  infection. 
A cell  is  never  entered  by  the  mycelium  until  it  has  ceased  to 
divide  and  until  it  has  reached  nearly  its  maximum  size.  An 
immediate  result  of  the  invasion  of  such  a cell  is  the  accumula- 
tion of  a large  amount  of  densely  staining  fine  granular  cyto- 
plasm as  is  shown  in  Figures  7 C and  G.  Soon  coarse  irregular 
shaped  solid  bodies  appear  which  are  like  those  described  by 
Magnus  and  Groom  and  assumed  by  them  to  bo  the  disintegrat- 
ing fungus.  Here,  however,  these  bodies  appear  while  the  my- 
celium is  intact  and  healthy.  These  clumps  are  plainly  the  pro- 
ducts of  the  host  cell;  perhaps  resulting  from  its  stimulated 
activity.  The  healthy  mycelium  can  often  be  found  passing 
through  empty  cells  in  close  proximity  to  cells  showing  these 
clumps.  The  bladders  are  often  of  such  size  o,r  number  that 
they  completely  fill  the  cells  leaving  no  trace  of  cell  contents. 
There  are  furthermore  fungus  filaments  which  give  direct  con- 
nection between  the  mycelium  and  the  bladders  in  different 
cells.  When  empty  the  thick  heavy  walls  of,  the  bladders  re- 
main for  the  most  part  rigid  and  intact. 

These  conditions  lead  one  to  the  conclusion  that  in  this  case 
the  intercalary  bladders  are  formed  at  the  expense  of  the  host 
cells  and  that  the  food  thus  stored  is  used  by  the  mycelium  in 
other  portions  of  the  host  tissue. 

From  the  histological  and  cvtological  studies  which  the  writer 
has  made  of  the  fungus,  it  appears  that  its  relation  to  the  in- 
dividual cells  of  the  roots  and  the  rhizomes  is  one  of  mild  para- 
sitism and  that  the  nearly  mature  cortical  cells  are  invaded 
and  the  cytoplasm  digested.  As  already  shown  the  fungus  is 
■still  more  destructive  in  the  leaves. 


242 


Wisconsin  Research  Bulletin  No.  18 


In  certain  areas  of  marsh  meadows  the,  Calamagrostis  plants 
do  not  harbor  the  fungus.  At  least  it  does  not  appear  on  the 
leaves  and  thus  far  the  study  made  of  the  roots  does  not  show 
its  presence  there.  The  best  development  of  the  grass  is  in 
these  fungus  free  areas. 

It  has  been  pointed  out  that  a large  number  of  culms  are 
killed  by  the  fungus  especially  in  the  early  spring.  The  effect  of 
this  is  marked  in  several  areas  that  have  exhibited  general  in- 
fection during  the  past  four  seasons.  In  areas  where  in  1908 
there  was  a vigorous  growth  of  Calamagrostis  with  considerable 
fungus  appearing  on  the  culms  and  in  which  the  appearance  of 
the  fungus  was  still  more  marked  in  1909  and  1910,  there  was  in 
1911  a marked  thinning  out  of  the  grass.  The  observations  in 
the  field  therefore  agree  with  the  conclusions  based  on  the  rela- 
tions found  in  the  roots:  viz.  that  the  fungus  is  perennial  in  the 
roots  with  injurious  effect. 

The  original  theory  of  Kamienski  (1881)  was  extended  by 
Frank  to  apply  to  many  more  cases  of  fungi  in  roots.  Frank 
(1885,  p.  33)  maintained  that  the  fungus  conducts  and  pre- 
pares food  for  the  host  and  especially  that  it  assimilates  humus 
compounds.  He  considered  that  even  purely  endotropic  myc-  1 
orrhizal  fungi  receive  food  from  external  sources  and  that  the- ; 
root  contained,  as  it  were,  a trapped  fungus,  the  host  ultimately j 
digesting  the  fungus  (1892  p.  267). 

Groom’s  view  (1895,  p.  354  and  356)  differs  in  that  he  con- 
siders that  the  fungus  in  Thismia  first  absorbs  food  from  the'; 
cells,  that  the  fungus  is  not  completely  digested  by  the  host 
cells,  and  that  in  the  outer  layers  it  “ actually  profits  by  the 
symbiosis”  (1895,  p.  356). 

Stahl  (1900)  argues  at  length  in  favor  of  the  view  that  both  j 
ectotropic  and  endotropic  mycorrhiza  furnish  supplies  of  elabo-  j 
rated  organic  compounds  from  without.  He  attempts  to  show ) 
that  the  host  plants  in  such  cases  are  weak  in  photosynthesis 
and  that  they  also  manifest  a decrease  of  ash  content  on  ac- ' 
count  of  this  weak  power  of  assimilation. 

In  the  case  of  Sclerotiuni  rhizodes  in  the  roots  of  C.  cana- 
densis it  is  clear  that  the  fungus  filaments  do  pass  out  into 
the  soil  where  there  is  reason  to  believe  that  they  can  thrive 
saprophytically.  It  is  possible  that  these  filaments  may  fur- 


A Sclerotium  Disease  of  Common  Grasses 


243 


nisli  the  endotropic  portions  with  various  mineral  salts  and 
organic  compounds  which  may  at. first  be  drawn  upon  by  the 
host  cells  thus  accounting  for  the  enlarged  cells  and  the  dense 
cytoplasm  found  in  spur  rootlets  during  the  early  stages  of 
infection.  Admitting  the  possibility  of  a benefit  of  this  sort,  it 
is  certain  that  the  ultimate  effect  of  the  fungus  both  on  indi- 
vidual cells  and  on  the  entire  plant  is  that  of  a parasite  es- 
pecially when  conditions  favor  the  development  of  the  fun- 
gus  on  the  leaves. 

There  have  been  numerous  investigators  who  have  noted  para- 
sitic behavior  in  various  myeorrhizal  fungi.  Reess  (1885)  studied 
Elaphomyces  on  pine  and  believed  that  the  fungus  obtains  food 
from  the  roots  of  the  pine.  Hartig  (1886)  opposed  Frank’s 
views  of  symbiosis  and  considered  that  the  fungi  in  question 
were  parasitic.  Nadson  (1908)  found  “mycorrhiza”  penetrat- 
ing roots  and  killing  large  numbers  of  oak  seedlings.  Boulet 
(1910)  reports  the  presence  of  endotropic  mycorrhiza  on  al- 
mond, apricot,  peach,  cherry,  plum,  apple,  pear,  etc.,  and  states 
that  the  fungus  lives  as  a parasite  but  still  has  a beneficial  effect 
on  the  host  except  when  it  attacks  the  roots  with  unusual  vigor. 

It  is  such  evidence  as  this  that  indicates  the  unsatisfactory 
state  of  our  knowledge  concerning  the  intimate  physiological 
relations  which  exist  when  a fungus  resides  as  is  the  case  in 
endotropic  mycorrhiza  within  living  plant  tissues.  After  all 
we  judge  parasitism  and  symbiosis  by  rather  gross  general  re- 
sults. 

Possibly  the  same  fungus  may  under  different  conditions 
manifest  varying  degrees  of  parasitism  or  of  symbiotic  re- 
lationship. A myeorrhizal  fungus  on  an  oak  may  be  at  one 
time  symbiotic  as  Frank  maintained  and  again  the  same  fun- 
gus may  be  parasitic  as  Nadson  reports. 

On  this  point  the  behavior  of  Sclerotium  rhizodes  is  espec- 
ially. interesting.  Judged  solely  by  its  behavior  in  the  roots 
it  might  be  considered  as  entering  into  symbiotic  relationship. 
At  any  . rate  it  is  not  here  apparently  seriously  parasitic.  It 
may  bring  into  the  root  various  mineral  and  organic  food 
substances  which  may  in  part  be  appropriated  by  the  root 
issues.  There  is  no  evidence,  however,  that  the  fungus  is  itself 
igested.  In  the  leaves,  however,  it  is  vigorously  parasitic, 
t the  time  when  the  culms  have  elongated  there  is  no  direct 


244 


Wisconsin  Research  Bulletin  No.  18 


connection  between  the  mycelium  in  the  leaves  and  that  in  the 
roots  and  it  can  not  he  considered  that  the  vigorous  develop- 
ment of  the  fungus  in  the  leaves  can  supply  the  underground 
mycelium  with  food.  The  mycelium  is  perennial  in  the 
roots  and  probably  in  the  soil,  but  is  rather  short-lived  in  the 
leaves.  The  vigor  of  the  parasitic  development  on  the  leaves 
varies  considerably  with  the  season  and  with  the  species  of 
host. 

It  is  certain  that  the  general  effects  of  the  fungus  on  the 
grasses  here  discussed  is  that  of  a parasite. 

Economic  Significance 

The  occurence  of  this  fungus  on  C.  canadensis  decreases  the 
yield  and  quality  of  the  hay  obtained  from  marsh  meadows. 
When  the  infection  is  as  high  as  47  per  cent,  as  was  the  case 
in  1911  on  the  marsh  meadow  most  carefully  studied,  this  loss 
is  considerable.  The  ability  of  the  fungus  to  infect  and  injure 
such  important  grasses  as  Phi  cum  pratense  and  Poa  pratensis 
is  also  a matter  of  economic  significance  although  the  injury  to 
these  two  grasses  is  not  serious  at  present,  so  far  as  observed. 

Cultivation  of  the  soil  and  crop  rotation  naturally  suggest 
themselves  as  a means  of  control  for  this  fungus.  This  would 
be  practicable  in  case  the  fungus  becomes  destructive  in  up- 
land meadows.  On  nearly  all  the  meadows  where  this  fungus 
has  been  found  tillage  is  now  impossible  and  until  improved 
drainage  makes  cultivation  possible  the  fungus  will  necessarily 
run  its  course.  The  observations  made  thus  far  indicate  that 
Agrostis  alia  is  not  attacked  and  if  this  be  true  the  introduc- 
tion of  this  grass  into  infected  marsh  meadows  might  be  advis- 
able. The  susceptibility  of  Calamagrostis  canadensis  to  at- 
tacks of  this  fungus  makes  the  use  of  this  species  on  semi-re- 
claimed marsh  lands  of  doubtful  value. 

Summary 

The  fungus  Sclerotium  rhizodes  attacks  the  leaves  of  various 
grasses  causing  them  to  become  dry,  rigid  and  bent  into  char- 
acteristic crooks.  Upon  the  leaves  appear  felts  of  mycelium 
from  which  sclerotia  develop. 

The  development  on  the  leaves  is  most  vigorous  during  April 
and  May  when  the  death  of  many  entire  culms  results. 


A Sclerotium  Disease  of  Common  Grasses  245 

At  Madison,  Wis.,  the  fungus  has  been  found  on  eleven  dif- 
ferent grasses:  Phalaris  arundinacea,  CalamagrosUs  neglecta, 
Poa  pratensis,  Pamcularia  nervata,  Phleum  pratense,  Hordeum 
jubatum,  Bromus  ciliatus,  Eatonia  pennsylvanica,  Agropyron 
caninum,  Agrostis  hy emails j and  CalamagrosUs  canadensis. 

It  is  especially  injurious  and  destructive  to  CalamagrosUs 
canadensis  which  serves  as  its  principal  host. 

The  fungus  is  vigorously  parasitic  in  the  leaves,  less  so  in  the 
buds  and  the  stems,  and  but  slightly  so  in  the  roots  where  it 
assumes  some  of  the  characters  usually  associated  with  mycor- 
rhiza. 

Fungus  filaments  extend  out  into  the  soil  where  they  live 
more  or  less  independently. 

The  mycelium  is  perennial  in  the  soil  and  in  the  underground 
parts  of  the  infected  plants. 

The  infection  of  aerial  parts  occurs  from  the  underground 
parts  of  the  plant. 

The  mycelium  is  sterile  so  far  as  has  been  observed  to  date. 

The  production  of  spore  bearing  structures  from  sclerotia 
has  never  been  observed. 

The  fungus  is  of  considerable  economic  importance.  It  de- 
stroyed or  dwarfed  as  many  as  47  per  cent  of  the  plants  of 
CalamagrosUs  canadensis  on  one  marsh  meadow  near  Madison 
in  the  season  of  1911. 

This  fungus  is  quite  generally  distributed  throughout  Wis- 
consin. It  has  not  been  reported  elsewhere  in  America;  pre- 
sumably it  is  quite  widely  distributed,  however.  Its  parasitic 
attacks  on  the  leaves  of  various  grasses  has  long  been  known 
in  Germany,  Belgium  and  Scandinavia. 

Further  data  are  desired  on  the  following  points:  additional 
host  plants,  geographical  range  of  the  fungus,  economic  im- 
portance, germination  of  the  sclerotia,  and  infection  of  seed- 
lings. 


246 


Wisconsin  Research  Bulletin  No.  18 


BIBLIOGRAPHY 

Arzberger  E.  G. 

1910.  Fungous  Root  Tubercles.  Ann.  Rpt.  Missouri  Bot. 
Garden  21 : 60-102. 

Atkinson,  G.  F. 

1893.  Symbiosis  in  the  Roots  of  the  Ophioglosseae.  Proe. 

Amer.  Assoc.  Adv.  Sci.  42;  254-255.  Also  in 
Bui.  Torrey  Bot.  Club  20:  356-357. 

Brefeld,  0. 

1881.  Untersuchungen  uebe,r  Schimmelpilze,  4. 

Boulet,  V. 

1910.  Sur  les  mycorhizes  endotrophes  de  quelques  arbes 
fruitiers.  Compt.  Rend.  Acad.  Sci.  [Paris]  150: 
1190-1192. 

Campbell,  D.  H. 

1908.  Symbiosis  in  Fern  Prothallia.  Amer.  Nat.  42 : 154-165. 
Davis,  J.  J. 

1893.  A Supplementary  List  of  the  Parasitic  Fungi  of 
Wisconsin.  Trans.  Wis.  Acad.  9:  153-188. 

Frank,  A.  B. 

1881.  Handbuch  der  Pflanzenkrankheiten. 

1885.  Neue  Mitteilungen  ueber  die  Mycorhiza  der  Baume 
und  der  Monotropa  liypopitys.  Ber.  Deut.  Bot. 
Gesell.  3:  XXVII— XXX. 

1887.  Sind  die  Wurzelanschwellungen  der  Erlen  und 
Elaeagnaceen  Pilzgallen?  Ber.  Deut.  Bot.  Gesell. 
5:  50-58. 

1887.  Ueber  neue  Myoorhiza-Formen.  Ber.  Deut.  Bot. 

Gesell.  5:  395-408. 

1892.  Lehrbuch  der  Botanik  1. 

1896.  Die  Krankheiten  der  Pflanzen. 

Freeman,  E.  M. 

1903.  The  Seed  Fungus  of  Lolium  femulentum  L., 
Darnel.  Phil.  Trans.  Roy.  Soc.  London  B 196: 
1-27. 

Groom,  P. 

1895.  On  Thismia  Aseroe  (Beccari)  and  its  Mycorhiza. 
Ann.  Bot.  9 : 327-361. 


A Sclerotium  Disease  of  Common  Grasses 


247 


Hartig  R. 

1886.  Ueber  der  symbiotischen  Erscheinungen  im  Pflan- 
zenleben.  Bot.  Centbl.  25 : 350-352. 

Hiltner,  L. 

1899.  Ueber  die  Assimilation  des  freien  atmospharischen 
Stickstoffs  dnrch  in  oberirdischen  Pflanzenteilen 
lebende  Mycelien.  Centbl.  Bakt.,  2.  5:  831-837. 

Janse,  J.  M. 

1896.  Les  endophytes  radicanx  de  quelques  plantes  java- 
naises.  Ann.  Jard.  Bot.  Bnitenzorg  14:  53-201. 

Kamienski,  F. 

1881.  Die  Vegetationsorgane  der  Monotropa  hypopitys  L. 

Bot.  Ztg.  39 : 457-461. 

1882.  Les  organes  vegetatifs  du  Monotropa  hypopitys  L. 

Mem.  Soc.  Sci.  Cherbourg  24 : 5-40. 

Lang,  W.  H. 

1899.  The  Prothallus  of  Lycopodium  clavatum.  Ann.  Bot. 

13:  279-316. 

Lntman,  B.  F. 

1910.  Some  Contributions  to  the  Life  History  and  Cyto- 
logy of  the  Smuts.  Trans.  Wis.  Acad.  16:  1191- 
1245. 

Magnus,  W. 

1900.  Studien  an  der  endotrophen  Mycor;rhiza  von  Neottia 

Nidus  avis  L.,  Jahrb.  Wiss.  Bot.  35:  205-272. 

Me  Alpine,  D. 

1910.  The  Smuts  of  Australia. 

Nadson,  G.  A. 

1908.  Zur  Lehre  von  der  Symbiose.  I,  Das  Absterben  von 
Eichensamlingen  im  Zusammenhange  mit  der 
Mycorrhiza.  Jah,rb.  Pflanzenkrankheiten.  St. 
Petersburg,  2 : 26-40.  Abst.  in  Centbl.  Bakt.  Abt. 
2,  26 : 100-101.  Exp.  Sta,  Record  22 : 722-723. 

Hemec,  B. 

1899.  Die  Mykorrhiza  einiger  Lebermoose.  Ber.  Deut.  Bot. 
Gesell.  17 : 311-316. 

Rees,  M. 

1885.  Ueber  Elaphomyces  und  sonstige  Wurzelpilze.  Ber. 
Deut.  Bot.  Gesell.  3:  293-295. 


248 


Wisconsin  Research  Bulletin  No.  18 


Saccardo,  P.  A. 

1899.  Sylloge  Fungorum.  14. 

Schlicht,  A. 

1889.  Beitrag  zur  Kenntniss  de,r  Verbreitung  und  der 
Bedeutung  der  Mykorhizen.  Inaug.  Diss.  Erlan- 
gen. 

Shibata,  K. 

1902.  Cytological  Stndien  neber  die  endotropben  Mycor- 
rhizen.  Jahrb.  Wiss.  Bot.  37:  643-684. 

Sorauer,  P. 

1886.  Handbuch  der  Pflanzenkrankheiten. 

Stahl,  E. 

1900.  De,r  Sinn  der  Mycorhizenbildung.  Jahrb.  Wiss  Bot 

34:  539-668. 

Stout,  A.  B.  A Statistical  Analysis  of  the  Vegetation  of  a 
Wild  Hay  Meadow.  To  be  published  in  Trans. 
Wis.  Acad.  17. 

Ternetz,  Charlotte. 

1907.  Ueber  de  assimilation  des  atmospharischen  Stick- 
stoffs  durch  Pilze.  Jahrb.  Wiss.  Bot.  44:  353 - 
408. 

Tubeuf,  K.  von. 

1897.  Diseases  of  Plants.  Eng.  Trans,  by  Smith. 

Woronin,  W. 

1885.  Ueber  dir  Pilzwurzel  von  B.  Frank.  Ber.  Deut.  BoU 
Gesell,  3:  205-206.  * 

Explanation  of  Figure  1 

This  photograph  shows  the  habit  of  growth  of  Calamagrostis  t 
canadensis  and  the  general  effects  of  the  fungus  Sclerotium 
rhizodes  as  it  appears  early  in  the  season.  Three  of  the  culms  ] 
here  shown  were  badly  infected,  the  leaves  were  shriveled  and 
rolled  especially  toward  the  tips,  the  characteristic  c,rooks  were 
present  and  the  felt  of  mycelium  can  be  seen  in  the  picture  at 
points  marked  m.  Young  sclerotia  were  present  at  points 
marked  s.  The  culm  which  stands  second  from  the  terminal 
bud  of  the  rhizome  had  escaped  infection. 

The  investigations  show  that  in  such  plants  as  this  the  fungus 
is  present  in  the  leaves,  stems,  buds,  and  roots  and  that  the 
mycelium  is  also  present  in  the  soil  and  on  the  roots. 


A Sclerotium  Disease  of  Common  Grasses 


249 


Explanation  of  Figure  2 

A life  sized  picture  of  a rapidly  growing  culm  of  Calama- 
grostis  canadensis  which  was  vigorously  attacked  by  the  fun- 
gus. The  appearance  of  the  crooks  and  their  method  of  forma- 
tion are  here  well  shown.  The  tips  of  the  unfolding  leaves  were 
penetrated  by  the  mycelium  which  held  them  together  in  a roll 
and  hence  as  the  leaves  developed  they  arched  upward.  Often 
the  growth  tensions  are  such  that  a portion  of  the  crook  is 
twisted  and  folded  as  is  the  case  here.  Death  of  the  entire 
culm  soon  results  from  such  a vigorous  development  of  the 
fungus. 

Explanation  of  Figure  3 

A.  Upper  portions  of  two  older  culms  of  Calamagrostis  cana- 
densis showing  conditions  somewhat  different  from  the 
previous  figures.  In  the  left  hand  figure  at  point 
marked  m is  seen  a partial  lateral  infection  of  a leaf. 
This  is  a common  phenomenon.  The  right  hand  figure 
shows  a complete  series  of  crooks,  five  in  number.  The 
tips  of  the  leaves  are  held  at  the  points  lettered.  These 
figures  illustrate  the  fact  that  every  leaf  of  an  infected 
culm  possesses  the  fungus  and  that  the  mycelium  gains 
entrance  to  the  leaves  when  they  are  in  the  bud.  While 
the  tips  of  the  leaves  here  shown  are  completely  de- 
stroyed there  is  a less  vigorous  development  of  the  fun- 
gus than  is  shown  in  Figure  2.  This  condition  is  seen 
during  the  middle  of  the  season,  especially  during  a 
rather  dry  period  when  few  sclerotia  are  formed.  Such 
culms  may  grow  for  some  time  but  seldom  produce 
flowers  and  seeds. 

B.  Portions  of  C.  canadensis  culms  and  leaves  showing  typical 
matured  sclerotia  as  they  are  produced  in  the  field  un- 
der favorable  conditions.  Culms  such  as  are  shown  at 
left  hand  side  soon  die  from  the  attack  of  the  fungus. 
(Natural  size) 

Explanation  of  Figure  4 

A.  A typical  culture  of  Sclerotium  rhizodes  on  hard  potato 
agar  showing  abundant  growth  of  mycelium  and  the 
crusted  pseudo-sclerotia.  Culture  is  ten  months  old. 


250 


Wisconsin  Research  Bulletin  No.  18 

B.  Culture  on  lima  bean  agar  nearly  ten  months  old.  The 

mycelium  is  less  abundant  than  in  A but  the  sclerotia 
are  more  perfect. 

C.  Development  of  the  fungus  on  bean  pods  with  a few  well 

formed  selerotia  and  abundant  mycelium. 

Explanation  op  Figure  5 

A.  Two  cultures,  15  months  old,  on  hard  potato  agar.  The 

mycelium  is  still  quite  vigorous  and  the  large  compound 
irregular  shaped  sclerotia  are  mature. 

B.  A pure  culture  of  the  fungus  on  peaty  marsh  soil  upon 

which  infected  Calamagrostis  canadensis  had  been 
growing.  The  soil  with  various  grass  roots  included 
was  sterilized  and  fragments  of  the  mycelium  from 
test  tube  cultures  were  placed  on  the  soil.  At  the  time 
the  photograph  was  taken  this  culture  was  ten  weeks 
old.  The  picture  shows  the  mass  of  white  mycelium 
that  covered  the  surface  while  toward  the  bottom  may 
be  seen  clusters  of  the  mycelium  which  also  spread 
quite  generally  through  the  soil  mass.  These  soil  cul- 
tures demonstrate  that  the  fungus  can  live  as  a sapro- 
phyte  in  the  soil. 

Explanation  of  Figure  6 

All  drawings  of  this  Figure  are  from  Calamagrostis 
canadensis. 

A.  Part  of  a cross  section  through  such  an  infected  leaf  roll  as 

is  shown  at  m,  Figure  3 A.  The  drawing  is  from  the 
outer  leaf  of  the  roll.  The  mesophyl  of  the  leaf  is  en- 
tirety destroyed.  (X  100). 

B.  Drawn  from  the  center  of  a badly  infected  roll  showing 

destruction  and  collapse  of  the  tissues  of  the  bundle. 

A and  B show  the  conditions  which  prevail  in  the  outer 
and  inner  portions  of  an  infected  roll  of  leaves 
(X  140). 

C.  From  the  same  cross  section  as  A,  but  from  the  border  of 

the  infected  area  showing  the  mycelium  advancing 
through  the  tissues.  (X  100), 


A Sclerotium  Disease  of  Common  Grasses 


251 


D.  Distribution  of  the  mycelium  about  the  growing  points  of 

a terminal  bud  of  a culm  the  expanded  leaves  of  which 
showed  infection  such  as  seen  in  Figure  1.  (X  100). 

E.  The  bud  from  which  D was  drawn.  Showing  the  distri- 

bution of  the  mycelium. 

F.  From  a cross  section  of  a stem  near  a node.  This  particular 

culm  was  two  feet  tall.  Its  leaves  were  infected.  A 
node  situated  immediately  below  the  surface  of  the 
ground  with  its  bud  was  fixed  during  the  first  week  in 
August.  At  that  time  the  culm  was  mature.  The  bud 
was  one  which  would  develop  during  the  next  year.  The 
mycelium  was  present  in  the  outer  layers  of  the  culm 
as  shown  in  the  segment  drawn.  Drawings  similar  to 
this  could  be  shown  for  the  various  nodes  of  infected 
culms.  (X  100). 

G.  Portion  of  the  bud  mentioned  above  drawn  in  its  relation 

to  the  stem  F and  from  the  same  cross  section.  The 
mycelium  is  present  in  the  tissues  of  the  bud  leaves  as 
well  as  between  the  leaves  where  it  remains  over  winter. 
(X  100). 

Explanation  of  Figure  7 

All  from  Calamagrostis  canadensis  unless  otherwise  stated. 

A.  Portion  of  a basal  node  showing  the  extent  of  penetration 

by  the  mycelium  and  the  vesicles  which  are  formed. 
(X  100). 

B.  Drawn  from  a longitudinal  section  through  a basal  node 

and  its  bud.  a is  a part  of  the  node  and  b is  from  the 
outside  leaf  of  the  bud.  The  mycelium  is  here  shown 
passing  from  stem  to  bud.  Material  was  collected  dur- 
ing the  summer.  (X  100). 

C.  From  a longitudinal  section  of  a fibrous  root.  Shows  con- 

dition of  cells  soon  after  penetration  by  the  mycelium ; 
the  nuclei  are  in  various  stages  of  degeneration  and 
there  is  an  accumulation  of  dense  material  in  the 
cytoplasm.  (X  140). 

D.  Cross  section  of  an  older  root  (the  main  root  of  Figure  8 

D)  showing  mycelium  and  vesicles  in  empty  cells. 
(X  100). 


252 


Wisconsin  Research  Bulletin  No.  18 


E.  Cross  section  of  a spur  o,r  lateral  root.  Shows  the  tendency 

of  the  fnngns  to  develop  in  the  innermost  layer  of  the 
cortex.  (X  140). 

F.  Mycelium  in  practically  empty  cortical  root  cells.  The 

densely  staining  granules  are  the  remains  of  the  proto- 
plasm of  the  host  cell.  Mycelium  is  intact.  (X  140).  I 

0.  From  a cross  section  of  a lateral  root  of  Calamagrostis 

neglecta.  Mycelium  is  here  seen  on  the  surface  of  the 
root,.  Formation  of  vesicles,  accumulation  of  dense 
protoplasm  and  abnormal  nuclei  are  here  shown 
(X  140). 

IT.  From  root  of  Poa  pratensis.  Mycelium  in  the  cortex 
(X  100). 

1.  Early  stages  of  vesicle  formation.  Nuclei  and  cytoplasm  of 

host  cells  nearly  disappeared.  Later  stages  are  shown 
in  Figure  8,  F and  G.  Figure  7,  C,  E,  F,  and  I,  and 
Figure  8,  F and  G present  a series  showing  the  stages 
in  the  development  of  the  mycelium  and  vesicles,  to- 
gether with  the  accompanying  disintegration  and  dis- 
appearance of  the  protoplasm  of  the  host  cell.  (X  140). 

J.  A few  cells  of  the  mycelium  grown  on  cooked  potato  show- 
ing the  prevailing  two-nucleated  condition.  (X  630). 

Explanation  of  Figure  8 

A.  Drawing  (X  140)  of  the  mycelium  as  it  develops  on  cooked 

potato.  The  oidium-like  cells  do  not  function  as 
spores,  but  are  similar  to  the  cells  in  a young  sclero- 
tium. 

B.  Characteristic  mycelium  from  the  surface  of  a leaf  of  C. 

canadensis  (X  140). 

C.  Drawing  (X  140)  of  mycelium  which  developed  from  the 

cut  end  of  an  infected  root.  i 

D.  A portion  of  an  infected  root  of  C.  canadensis  is  here  shown 

(X  10)  with  the  enlarged  spar  rootlets  and  the  myce- 
lium as  it  exists  in  the  soil  and  on  the  surface  of  the 
roots.  The  large  bladder-like  vesicles  as  they  appear 
in  the  soil  are  here  shown. 

E.  A small  portion  of  a rootlet  (X  100).  The  mycelium  is  here 

shown  adhering  to  the  surface  of  the  rootlet  and  pene- 
trating  into  the  root  tissues  at  two  points. 


A Sclerotium  Disease  of  Common  Grasses 


253 


T.  Typical  vesicles  as  they  appear  within  cells  of  the  cortex 
of  roots  of  C.  canadensis  (X  175). 

G.  A single  spherical  vesicle  within  a cell  of  a root  of  C. 

neglecta  (X  175). 

H.  Diagrammatic  cross  section  of  a leaf  which  is  laterally  in- 

fected. The  dark  portion  represents  the  distribution 
of  the  mycelium.  The  tip  of  a second  leaf  is  shown 
within  the  roll. 


Wisconsin  Research  Bulletin  No.  18 


254 


Figure  1.  C.  canadensis,  showing  tlie  general  symptoms  of  the  Sclerotium 

disease. 


A Sclerotium  Disease  of  Common  Grasses 


Figure  2.  The  characteristic  “crooks”  resulting  from  a vigorous  attack  of  the 

Sclerotium 


\ 


256 


Wisconsin  Research  Bulletin  No.  18 


Figure 


Illustrations  of  various  leaf  symptoms  associated 
tium  disease 


with  the  Sclero- 


A Sclerotium  Disease  of  Common  Grasses 


257 


ABC 

Figure  4.  Pure  cultures  of  Sclerotium  rliizodes  on  various  media 


Wisconsin  Research  Bulletin  No.  18 


258 


A 

Cultures  of  the  Sclerotium  ; A showing  the  large  sclerotia  ; B the 
mycelium  in  soil 


A Sclerotium  Disease  of  Common  Grasses 


259 


Figure  6.  The  mode  of  tissue  invasion  of  grass  leaves  and  stems  by  the  Sclero- 


tium mycelium 


260 


Wisconsin  Research  Bulletin  No.  18 


Figure  7.  A— H,  Development  of  the  Sclerotium  in  the  tissues  of  leaf,  node;  and 

root;  J,  mycelium  from  culture 


A Sclerotium  Disease  of  Common  Grasses 


261 


Figure  8.  A-C,  mycelium  of  the  sclerotium;  D-G,  infected  roots;  H,  infected  leaf 


'^XM^MsChs  bviJLfcAA, 

**■  ie 

Effect  of  Heat  and  Oxidation  on  the 
Phosphorus  of  the  Soil 


BY  P.  P.  PETERSON 


Prefatory  Note 

It  is  a well  known  fact  that  virgin  soils,  as  a rule,  show  a 
high  degree  of  fertility.  This  fertility  lasts  for  a variable 
length  of  time  but  in  practically  all  cases  when  exhaustion  does 
come,  it  is  phosphorus  alone  or  with  other  elements  which  has 
become  lacking  to  the  crop.  Nevertheless,  when  a determina- 
tion of  the  total  amount  of  phosphorus  in  the  virgin  and  crop- 
ped soils  is  made,  it  is  always  found  that  only  a part,  and  often 
a small  part,  of  the  amount  present  in  the  virgin  condition 
has  been  removed  before  the  soil  shows  exhaustion.  This  is 
particularly  true  of  soils  having  a large  amount  of  organic  mat-  „ 
ter  such  as  marsh  and  black  prairie  soils.  These  facts  suggest 
that  the  phcfsphorus  in  virgin  soils  which  becomes  available 
readily  is  in  organic  matter  which  is  easily  oxidized.  To  test  this 
hypothesis  Dr.  A.  F.  McLeod,  formerly  of  this  Department,  at 
the  suggestion  of  the  writer  undertook  the  oxidation  of  virgin 
soils  by  the  use  of  varying  strengths  of  hydrogen  peroxide  with 
the  thought  that  it  would  be  possible  by  so  doing  to  extract  the 
phosphorus  set  free  in  such  oxidation.  It  was  found,  however, 
that  fixation  occurred  so  rapidly  that  the  phosphorus  set  free 
by  oxidation  could  not  be  extracted.  The  investigation  was 
later  taken  up  by  Dr.  P.  P.  Peterson,  and  the  results  reported 
in  the  following  pages  secured. 


A.  R.  Whitson. 


2 


Wisconsin  Research  Bulletin  No.  19 


Introduction 

The  important  role  which  the  phosphorus  of  the  soil  plays  in 
crop  production  has  long  been  known.  In  1804  De  Saussurer 
speaking  of  calcium  phosphate  which  he  had  found  in  the- 
ashes  of  plants,  wrote  as  follows:  “I  have  found  this 

same  salt  in  the  ashes  of  all  plants  that,  I have  investigated,  and 
there  is  no  reason  to  assert  that  they  could  exist  without  it.,r 
But  little  was  written  on  the  subject  until  about  1840  when 
Liebig  and  his  contemporaries  took  it  up  with  renewed  interest, 
and  from  that  time  the  interest  has  been  kept  up.  As  a result 
the  complexity  of  the  -problem  and  the  literature  have  grown 
apace.  The  question  that  De  Saussure  raised  in  the  statement 
quoted  above  was  a simple  one  and  was  soon  settled  in  the  affir- 
mative. Plants,  in  general,  do  need  phosphorus  for  their  best 
growth.  Boussingault  took  up  the  next  question : ‘ ‘ How  much 
phosphorus  does  a crop  remove  from  the  soil?”  It  was  soon 
discovered  that  different  kinds  of  crops  use  different  amounts  of 
phosphorus.  Magnus  attempted  to  show  that  phosphorus  is  not 
a limiting  factor  in  crop  production.  He  showed  that  a crop  of 
turnips  removes  so  small  an  amount  of  phosphorus  from  the  soil 
as  to  make  it  possible  for  them  to  be  grown  almost  indefinitely 
on  the  same  soil  without  exhausting  the  supply  of  phosphorus. 
Liebig  took  up  the  other  side  of  the  question  and  soon  won  the 
approval  of  the  chemists  by  his  arguments. 

Among  the  questions  which  have  arisen  since  the  time  of  Lie- 
big is  that  of  the  availability  of  the  phosphorus  of  the  soil  to 
plants.  Various  methods  have  been  devised  for  measuring  the 
availability  but  none  are  thoroughly  reliable.  They  give  only  a 
rough  approximation.  The  one  that  has  been  used  in  this  work 
is  that  developed  by  Stoddart,1  the  fifth  normal  nitric  acid  ex- 
traction method.  While  the  theory  upon  which  this  method  is 
based  is  doubtless  faulty,  it  has  been  shown  practically  that 
when  the  solubility  of  phosphoric  anhydride  in  fifth  normal 
nitric  acid  is  lower  than  .015  per  cent  the  soil  is  likely  to  be 
deficient  in  available  phosphorus.  And  since  in  this  work  an 
approximation  is  the  best  that  can  be  expected,  this  method  has 
been  suitable. 


i Jour,  of  Ind.  & Eng.  Chem.  1,  No.  2,  p.  7 


Effect  of  Heat  and  Oxy< 


gen  on  Soil  Phosphorus  3 


In  Wisconsin  there  a, re  many  soils  high  in  total  phosphorus 
and  yet  deficient  in  available  phosphorus  when  measured  by  its 
solubility  in  fifth  normal  nitric  acid.  Table  I gives  data  for  a 

number  of  soils  with  which  we  have  worked  that  show  this 
characteristic. 


Table  I 


Some  Wisconsin  Soils  High  in  Total  Phosphorus 
in  Available  Phosphorus 


but  Low 


Lab. 

No. 


Kind  of  Soil 


Location 


P2O5  Sol- 
uble 

in  N/5HNO3 
Per  cent  of 
soil 


Total  P2O5 
Per  cent  of 
soil 


234 

239 

283 

296 

485 

519 

1288 

1371 

3X 


Virgin 

Cropped,  well  handled!.. 

Virgin 

Virgin 

Cropped,  still  considered  to  be 
in  g-ood  state  of  fertility 

Virgin 

Cropped  (a)  ” 

Virgin 

Cropped  (a)...!! !!!!!! 


St.  Croix  Co. 
Vernon  Co. . . 

Grant  Co 

Richland  Co. 


.013 

.008 

.009 

.010 


.117 

.154 

.175 

.149 


Watertown.., . . . 
Washington  Co. 

Richland  Co 

Waukesha  Oo. . . 
Jefferson  Co 


.014 

.012 

.003 

.013 

.013 


.139 

.187 

.204 

.121 

.067 


In  considering  this  table,  the  following  things  should  be 
noted.  The  soils  have  come  from  various  parts  of  the  state. 
Of  the  nine,  five  are  virgin  and  four  cropped.  Therefore  these 
do  not  represent  a class  of  soils  that  have  been  exhausted  by 
cropping.  They  have  not  been  selected  because  of  their  acid  or 
non-acid  character. 

The  non-availability  of  so  large  a part  of  the  total  phosphorus 
of  the  soil  caused  us  to  carry  on  an  investigation  to  deter- 
mine if  possible  in  what  condition  it  existed  to  make  it  so  use- 
less to  plants.  There  are  many  compounds  of  phosphorus  whieh 
may  be  present  in  the  soil.  Some  are  inorganic  and  doubtless 
•some  organic  compounds  also  are  there.  It  is  possible  also  that 
oth  phosphorus  and  iron  or  aluminum  may  be  in  combination 
m the  same  organic  molecule.  Eggertz,2  working  with  the  am- 
moniacal  extract  of  a soil,  found  that  it  contained  from  .15  to 
7.58  per  cent  of  phosphorus  and  concluded  that  the  phosphorus 
was  chemically  combined  with  carbon.  Van  Bemellen3  found 
a phenomenon  of  absorption  of  phosphorus  by  the  soil  and  be- 
lieved  that  the  phosphorus  not  chemically  combined  in  minerals 


2Centbl.  Agr.  C'hem.  18,  75 
3 Landw.  Vers.  Stat.  37,  347 


4 


Wisconsin  Research  Bulletin  No.  19 


was  absorbed  physically  by  a humate-silicate  colloid,  but,  not  in 
chemical  combination  with  carbon.  Wikhind 4 5 brought  evi- 
dence against  Van  Bemellen  and  Schmoeger3  followed  with  his 
classical  work,  confirming  Eggertz  and  Wiklund  and  concluding 
also  that  the  organically  bound  phosphorus  consisted  mainly  of 
nuclein.  This  conclusion  he  arrived  at  by  heating  a peat  soil 
to  140° — 160°  C in  an  autoclave.  This  gave  a large  increase  in 
phosphorus  soluble  in  12  per  cent  hydrochloric  acid.  Nuclein 
when  heated  in  the  same  way  splits  off  phosphoric  acid.  He 
also  found  a similar  increase  in  the  solubility  of  sulphur  in 
both  soil  and  nuclein  after  heating.  This  similarity  between 
the  soil  phosphorus  and  soil  sulphur  on  the  one  hand  and 
nuclein  phosphorus  and  nuclein  sulphur  on  the  other  he  inter- 
preted to  mean  that  the  phosphorus  was  combined  with  organic 
matter  in  the  form  of  nuclein.  Aso6  did  the  same  experiments 
with  a peat  front  Japan  and  found  the  same  results  except  that 
the  increase  in  sulphur  obtained  by  heating  the  peat  was  not 
nearly  so  marked.  They  both  found  some  lecithin  in  the  peat  but 
fhe  amount  was  inconsiderable. 

The  method  which  Grandeau  developed  for  the  extraction  of  ■ 
organic  matter  from  the  soil  has  been  used  to  estimate  the  or-  * 
ganically  bound  phosphorus.  The  precipitate  which  is  thrown  \ 
down  by  adding  a strong  acid  to  the  ammonia  solution  contains  ! 
phosphorus.  Grandeau 7 considered  this  to  be  from  organic , 
sources.  Nannes  8 showed  that  the  filtrate  also  contained  pnos-  j 
phorus.  Out  of  the  .166  per  cent  contained  in  the  peat 
with  which  he  worked,  .057  per  cent  was  found  in  the 
matiere  noire,  .039  per  cent  was  in  the  filtrate.  Ladd 9 es- . 
timated  that  on  an  average  nearly  half  of  the  phosphorus  of . 
eight  soils  with  which  he  worked  was  in  organic  form  though 
this  varied  greatly  for  the  individual  soils,  A question  has' 
been  raised  by  F.raps  as  to  the  correctness  of  the  conclusions 
-of  Nannes  and  Ladd.  Fraps10  finds  that  four  per  cent 
ammonia  will  dissolve  aluminum  and  iron  phosphates  and  thinks 
part  of  the  phosphorus  obtained  by  the  Grandeau  extraction 


4 Landw.  Jahrb.  20,  909 

5 Ber.  der  Deutsch.  Chem.  Ges.  26,  336 

g Tokyo  Col.  of  Agr.  Bui.  6,  277. 

v Compt.  Rend.  98,  201 

s Jahresberechte  liber  Agrikulturcbemie  42,  89 

9 N.  Dak.  Exp.  Sta.  Bui.  32,  310 

10  Am.  Chem.  Jour.  39,  204 


Effect  of  Heat  and  Oxygen  on  Soil  Phosphorus  5 

is  from  this  source.  In  a recent  work11  he  estimated  that 
in  twenty-seven  of  the  soils  with  which  he  worked,  51  per  cent 
of  the  phosphorus  extracted  by  4 per  cent  ammonia  after  a treat- 
ment with  12  per  cent  hydrochloric  acid  is  from  inorganic 
sources. 

Hopkins  and  Petite12  suggest  a method  for  estimating  the  or- 
ganic phosphorus  of  a soil  by  the  difference  in  total  phosphorus 
in  the.  surface  and  subsoil.  Stewart,13  working  at  the  Illinois 
Experiment  Station,  brought  confirmatory  evidence  of  their 
view  by  comparing  the  amount  of  phosphorus  in  the  organic 
form  as  determined  by  the  various  methods.  In  his  work  he 
found  that  by  the  method  of  calculation  of  Hopkins  and  Petite, 
46  per  cent  of  the  total  phosphorus  was  in  organic  combination' 
By  the  method  of  Nannes  and  Ladd  he  found  55  per  cent,  by 
the  method  of  ignition  60  per  cent  and  by  the  autoclave  method 
of  Schmoeger  66  per  cent.  Fraps  points  out  the  errors  likely 
to  influence  the  results  in  the  three  methods  last  named.  In  the 
cases  of  the  method  of  Nannes  and  Ladd  and  the  ignition 
method,  he  brought  experimental  evidence  against  them,  but  this 
is  not  true  of  the  autoclave  method.  Beside  the  objections 
raised  by  Fraps  to  the  conclusions  of  Stewart  there  seems  to 
be  another  objection  to  the  method  of  Hopkins  and  Petite.  In 
many  soils  the  difference  in  the  total  phosphorus  is  in  favor  of 
the  subsoil.  This  is  true  in  some  of  the  soils  with  which  they 
were  working  when  they  suggested  the  method.  For  these 
soils  the  method  certainly  can  not  be  applicable.  We  must  con- 
clude therefore  that  the  method  is  not  reliable. 

The  "ignition  soluble’7  method  was  used  by  Nagoaka.14  He 
tried  the  solubility  of  the  phosphoric  acid  in  strong  hydrochloric 
acid,  water,  ammonium  nitrate,  5 per  cent  acetic  acid,  1 per  cent 
citric  acid,  1 per  cent  oxalic  acid,  and  a saturated  solution  of 
carbonic  acid.  In  all  except  the  water  in  which  only  a trace 
was  soluble,  he  found  a material  increase  in  solubility  by  heat- 
ing. He.  also 15  compared  the  increase  in  solubility  of  phos- 
phoric acid  obtained  by  ignition  by  autoclave  heating  at  a pres- 
sure of  three  atmospheres,  and  by  steaming  in  Koch’s  apparatus. 

11  Tex.  Sta.  Bui.  136,  24 

12  111.  Sta.  Bui.  125,  204 

13  111.  Sta.  Bui.  145 

14  Tokyo  Col.  of  Agr.  Bui.  4,  265 

15  Tokyo  Col.  of  Agr.  Bui.  4,  265 


6 Wisconsin  Research  Bulletin  No.  19 

He  found  that  autoclave  heating  gives  a greater  increase  in 
the  solubility  than  either  of  the  others.  Steaming  in  Koch’s 
apparatus  gave  practically  no  increase.  Stewart 16  compared 
this  method  with  other  methods  and  found  a smaller  increase 
by  it  than  by  the  autoclave  heating  method.  Fraps17  criti- 
cizes the  ignition  method  because  ignition  causes  an  increase  in 
the  solubility  of  the  mineral  phosphates  wdiich  may  be  in  the  soil. 
The  assertion  that  the  phosphorus  may  come  from  minerals  is 
certainly  well  founded. 

Konig,  Hasenbaumer  and  Grossmann18  showed  that  phos- 
phoric acid  was  made  more  soluble  in  pure  and  carbonic  water 
by  oxidizing  the  soil  with  hydrogen  peroxide.  They  were  able 
to  destroy  70  per  cent  of  the  organic  matter  of  the  soil  in  this 
way  using  very  strong  hydrogen  peroxide.  McLeod  19  working 
in  this  laboratory,  showed  that  most  of  the  organic  matter  could 
be  removed  from  soils  by  a comparatively  weak  solution  of  hy- 
drogen peroxide.  In  the  investigation  reported  in  the  following 
pages  this  method  of  decomposing  the  organic  matter  has  been 
used  together  with  a method  of  dry  heat,  also  used  by  Konig. 
The  increase  in  the  solubility  of  the  phosphoric  acid  by  heating 
the  soil  may  come  from  the  mineral  phosphates  as  Fraps20  has 
pointed  out,  but  the  increase  by  oxidation  can  hardly  be  from 
that  source,  except  it  should  be  by  removing  from  the  mineral 
particles  an  envelope  which  protects  them  from  the  solvents. 
This  possibility  will  be  considered  in  the  experimental  part  of 
the  bulletin. 

EXPERIMENTAL  PART 

Solvent  Action  of  N/5  Nitric  Acid  on  Wavellite  and  Du- 
frenite  Before  and  After  Heating 

The  amount  of  phosphoric  acid  extracted  by  fifth  normal  ni- 
tric acid  on  raw  and  roasted  w^avellite  and  dufrenite  was  first 
tried  to  determine  if  the  effect  that  Konig  had  found  on  the 
solubility  of  the  phosphoric  acid  of  the  soil  by  dry  heat  could  be 
due  to  the  presence  of  these  minerals  in  the  soil.  A sample  of 

i«  111.  Exp.  Sta.  Bui.  145 
i7  Tex.  Sta.  Bui.  136,  29 
is  Landw.  Vers.-Stat.  69,  30 
is  Unpublished  work 
20  Am.  Chem.  Jour.  39,  204 


Effect  of  Heat  and  Oxygen  on  Soil  Phosphorus  7 


<eacli  weighing  2.5  grams  was  heated  in  a drying  oven  to  the 
temperature  given  in  Table  II.  Each  sample  was  then  digested 
with  250  c.  c.  of  fifth  normal  nitric  acid  and  100  c.  c.  taken  for 
duplicate  determinations  of  phosphoric  acid,  (P  2 05).21  Ta- 
ble II  shows  that  the  phosphoric  acid  of  wavellite  is  more  solu- 


Table  II  Effect  of  Heat  on  the  Solubility  of  Wavellite  and 

Dufrenite 


Temp.  C. 

Percent  of  Total  P2O5 
Soluble  in  N/5  HNO3 

W a vellite 

Dufrenite 

Not  heated 

4.12 

54.9 

49.0 

98.7 

0.8 

160° 

200° 

1.08 

240° 

ble  after  roasting  than  before.  Dufrenite  is  only  slightly  in- 
creased in  solubility  at  a temperature  of  200°.  The  wavellite 
contained  18.79  per  cent  and  the  dufrenite  19.6  per  cent  of 
phosphoric  acid. 


Solvent  Action  of  N/5  Nitric  Acid  on  the  Phosphoric  Acid 
of  the  Soil  Before  and  After  Heating 


For  the  experiment  with  soils,  25  grams  was  digested  with 
2o0  c.  c.  of  fifth  normal  nitric  acid  and  100  c.  c.  of  the  extract 
taken  fo,r  each  of  duplicate  determinations  of  phosphoric  acid. 
Because  of  the  increased  solubility  of  the  organic  matter  after 
heating  to  a high  temperature  the  extracts  of  the  samples  of 
soil  heated  above  130°  were  a dark  brown.  The  organic  matter 
was  destroyed  by  treatment  with  bromine  in  alkaline  solution 
until  the  residue  left  on  evaporation  was  a pure  white.  After 
heating  the  residue  to  120°  C.  for  several  hours  to  dehydrate  the 
silica  it  was  taken  up  with  nitric  acid  and  the  phosphoric  acid 
determined  in  the  usual  way.  To  avoid  oxidation  of  the  organic 
matter  which  might  take  place  by  the  atmospheric  oxygen  at 
high  temperatures  the  soil  was  heated  in  a vacuum  obtained  by 
a filter  pump. 

This  operation  was  carried  out  as  follows:  The  soil  was 

placed  in  a round  bottom  flask  stoppered  by  a one-hole  rubber 


21  Throughout  this  article  the  term  “phosphoric  acid” 
‘phosphorus  pentoxide.” 


will  used  for 


8 Wisconsin  Research  Bulletin  No.  19 

stopper  through  which  passed  a glass  tube  connected  with  a man- 
ometer  and  the  filter  pump.  The  pressure  as  read  on  the  mano- 
meter varied  between  10  and  15  mm.  Heating  was  accomplished 
at  temperatures  up  to  100°  C.  by  means  of  a water  bath  and  for 
the  higher  temperatures  by  a Rose  ntetal  bath.  A clay  loam  soil 
from  the  same  farm  as  1288  and  closely  corresponding  to  it 
except  in  total  phosphoric  acid  was  used.  The  total  phosphoric 
acid  was  .154  per  cent.  Table  III  gives  the  results  obtained. 


Table  III  Effect  of  Heat  on  the  Solubility  of  the  Phosphoric 

Acid  of  the  Soil 


Temp.  C. 

P2O5  soluble  in  N/5  HNO3 
Per  cent  of  soil 

.0028 

.0032 

.0038 

.0073 

.0178 

.0550 

.0440 

It  should  be  noticed  that  heat,  has  little  effect  upon  the  solu- 
bility of  the  phosphoric  acid  until  a temperature  considerably 
above  100°,  the  ordinary  cooking  temperature,  has  been  reached. 
A comparison  of  Tables  II  and  III  shows  a lack  of  similarity 
in  the  effect  of  heat  upon  wavellite  and  the  phosphoric  acid  of 
the  soil.  With  both,  an  increase  takes  place  at  about  160°,  but 
this  is  not  so  marked  in  the  soil  as  in  wavellite.  At  200°  the 
difference  is  in  the  other  direction.  The  solubility  of  the  soil 
phosphorus  reaches  its  maximum  at  this  temperature,  whereas 
the  solubility  of  the  wavellite  is  no  higher  than  in  that  heated 
to  only  160°.  At  240°  the  wavellite  becomes  almost  totally  solu- 
ble. 

Effect  of  Oxidation  with  Hydrogen  Peroxide  on  the  Sol- 
ubility of  the  Phosphoric  Acid  of  the  Soil  in  N/5 
Nitric  Acid 

While  there  is  this  lack  of  similarity  between  the  action  of 
heat  upon  wavellite  phosphoric  acid  and  soil  phosphoric  acid, 
it  is  seen  that  the  idea  of  Fraps  22  that  the  phosphoric  acid  comes 


22  Am.  Chem.  Jour.  39,  579 


Effect  of  Heat  and  Oxygen  on  Soil  Phosphorus 

from  the  minerals  still  is  very  probable.  But  we  have  to  recon- 
cile this  idea  with  the  experimental  results  of  Kbnig,  Hasen- 
baumer,  and  Grossmann,23  that  oxidation  by  hydrogen  peroxide 
sets  free  phosphoric  acid  from  the  soil.  It  is  hardly  to  he  ex- 
pected that  oxidation  can  set  free  phosphoric  acid  from  the 
minerals.  If  it  cannot  do  so  and  there  are  iron  and  aluminum 
phosphates  in  the  soil,  a second  increase  should  take  place  if  the 
soil  is  heated  after  the  organic  matter  has  been  destroyed  by 
hydrogen  peroxide.  Experiments  were  carried  out  to  determine 
whether  this  was  the  case  or  not.  Fifty  grams  of  soil  was 
weighed  into  a tared  beaker,  wet  with  50  c.  c.  of  water,  and  5 
c.  c.  of  a 30  per  cent  solution  of  hydrogen  peroxide  added.  The 
beaker  was  covered  with  a watch  crystal  to  prevent  loss  by  spat- 
tering. After  the  hydrogen  peroxide  was  exhausted,  5 c.  c.  more 
was  added  until  effervescence  ceased.  Then  the  beaker  was 
placed  on  a boiling  water  bath  when  the  reaction  began  again. 
This  treatment  was  continued  until  the  soil  ceased  to  lose  weight 
on  successive  additions  of  hydrogen  peroxide  and  drying  to 
constant  weight  at  100°.  The  difference  between  the  weight 
of  the  beaker  and  soil  at  the  beginning  and  end  of  the  opera- 
tion gave  the  loss  in  organic  matter.  The  soil  was  then  pulver- 
ized and  thoroughly  mixed  and  divided  into  equal  portions.  One 
portion  was  heated  to  240°,  the  other  was  not.  A third  sample 
of  25  grams  was  heated  without  previous  oxidation  and 
a blank  sample  of  the  same  weight  was  prepared  by  wetting  and 
drying  it  just  as  the  oxidized  sample  had  been  wet  and  dried. 
The  four  samples  thus  prepared  were  extracted  with  fifth  normal 
nitric  acid  in  the  usual  way.  The  extract  was  heated  with  bro- 
mine water  to  decompose  the  dissolved  organic  matter,  and  the 
phosphoric  acid  determined  in  aliquot  parts  of  100  c.  c. 

Soil  No.  1286  is  a virgin  clay  loam  soil  from  Gotham.  No. 
1288  is  a cropped  sample  corresponding  closely  to  1286  (See 
Table  I.).  No.  1363  is  a cropped  soil  from  a field  adjoining 
the  one  from  which  1288  was  taken.  Until  about  six  years  ago 
it  was  allowed  to  run  down,  but  since  that  time  has  been  well 
handled.  No.  1175  is  a cr6pped  soil  from  Pierce  County.  It  is 
a sandy  loam  rather  low  in  total  phosphoric  acid  but  high,  in 
available  phosphoric  acid.  No.  1176  is  a virgin  sample  corre- 
sponding very  closely  to  1175.  No.  3x  was  taken  from  a barrel 


23  Landw.  Vers.-Stat.  69,  30 


10 


Wisconsin  Research  Bulletin  No.  19 


sample  from  Ft.  Atkinson.  It  is  a crop  soil  which  responded 
to  a pot  test  for  phosphoric  acid.  No.  1365  is  a virgin  clay 
loam  from  Brookfield.  No.  1371  is  a virgin  sandy  loam  from 
Lenesee.  No.  1374  is  a virgin  sand  from  Eagle. 

Table  IV  gives  the  results  obtained  in  this  experiment. 


Table  IV  Solubility  op  P8Os  in  N/5  HNOa  Before  and  After  Oxi- 
DIZING  AND  HEATING  TO  230°  C 


6 

& 

JD 

«S 

Total 
P2O5 
Per  cent 
of  soil  (a) 

1 

1286 

.231 

1288 

.204 

1364 

.159 

1175 

.095 

1176 

.118 

3x 

.067 

1365 

.105 

1371 

.121 

1374 

.069  1 

1 

P2O5  Extracted  by  N/5  HNO 
of  Soil 

» Per  Cent 

Total  or- 

Before 

treatment 

After 
heating  to 
230°  C 

After  oxi- 
dizing 
with  H2O2 

After  oxi- 
dizing and 
1 then  heat- 
ing to  230° C 

ganic  mat- 
ter. 

Per  cent 
of  soil  | 

.012 

.003 

.004 

.024 

.034 

.013 

.020 

.096 

.059 

.060 

.050 

.066 

.056 

.130 

.090  1 

.068 
.059 
.074 
.062 
.064 

.124 

.079 

.062 

.051 

.072 

.062 

.057 

.047 

.035 

5.98 

3.54  I 

4.54 
1.42 
2.57 

2.35  ! 

.013 

.043 

.012 

.033 

1 

Organic 
matter  re- 
moved by 
H2O2 
Per  cent 
of  soil 


3.44 

1.32 

2.26 

2.08 

2.54 

1.38 

0.60 


(a)  Determined  by  fusion  with  Na2  C03. 


In  this  table  there  are  several  things  that  are  worthy  of  note. 
In  the  cases  where  the  data  are  sufficient  to  calculate  the  per- 
centage of  the  organic  matter  removed  by  hydrogen  peroxide, 
on  an  average,  nearly  90  per  cent  of  the  organic  matter  has  been 
removed.  Kbnig  was  able  to  remove  only  70  per  cent  with  a 
much  stronger  solution  of  hydrogen  peroxide.  A comparison  of 
columns  3 and  4 shows  that  a very  large  increase  in  the  solu- 
ble phosphoric  acid  is  obtained  by  heat  alone.  But  the  differ- 
ences between  the  figures  in  3 and  4 are  considerably  smaller 
m every  case  than  the  differences  between  the  figures  in  3 and  5 
which  represent  the  increases  obtained  by  oxidation  alone.  Per- 
haps the  most  notable  thing  in  this  table  is  that  it  shows  for 
most  of  the  soils  a decrease  in  the  solubility  of  the  phosphoric 
acid  rather  than  an  increase  on  heating  subsequent  to  oxidation. 
In  only  two  cases  is  an  increase  shown  and  they  are  so  small 
as  to  be  within  the  limit  of  experimental  error.  Where  a de- 
crease is  shown,  it  too  is  within  the  limit  of  experimental  error 
However,  a decrease  might  be  expected,  for  if  ferric  or  alumi- 
num hydrate  or  ferric  or  aluminum  oxide  is  present  it  will  be- 


Effect  of  Heat  and  Oxygen  on  Soil  Phosphorus  11 

come  much  less  easily  soluble  after  being  heated  to  230°  than  it 
was  before.  It  is  then  likely  to  hold  occluded  small  particles  of 
ferric  or  aluminum  phosphate  and  thus  prevent  it  from  the  action 
of  the  solvent.  The  sandy  soils  do  not  show  as  large  an  increase 
in  the  solubility  of  the  phosphoric  acid  as  the  clays  do, 
perhaps  because  of  the  smaller  amount  of  organic  matter  which 
they  contain.  The  average  increase  for  fine  clay  and  clay  loam 
-soils  is  50  per  cent  of  the  total  phosphorus.  For  the  sands  and 
sandy  loams  it  is  31  per  cent. 

The  fact  that  there  is  no  increase  in  the  solubility  of  the 
phosphoric  acid  on  heating  a soil  from  which  the  organic  matter 
has  been  removed  is  of  special  interest  here  because  of  Fraps’ 
work  in  which  he  concludes  that  much  of  the  “ignition  soluble’7 
tmd  1 1 ammonia  soluble”  phosphoric  acid  comes  from  the  mineral 
phosphates  of  aluminum  and  iron.  We  cannot  say  positively 
yet  that  there  are  no  mineral  phosphates  in  the  soil  to  account 
for  the  increase  in  solubility  of  phosphorus  on  heating  the  soil, 
but  the  results  point  in  that  direction.  In  the  soils  with  which 
we  have  worked  there  are  no  mineral  phosphates  which  increase 
in  solubility  upon  being  heated  to  230  D.  Before  a broader  con- 
clusion can  be  drawn,  further  work  will  have  to  be  done  with  a 
more  varied  class  of  soils  and  with  other  solvents. 

Increase  in  Soluble  Phosphoric  Acid  at  Different  Stages 

of  Oxidation 

From  the  facts  at  hand  we  could  not  be  certain  that  all  of 
the  phosphorus  held  by  the  organic  matter  was  released  by  the 
oxidation  as  carried  out.  To  make  this  point  more  certain  and 
to  discover  in  what  stage  of  oxidation  the  greater  part  of  the 
phosphorus  is  set  free,  experiments  were  carried  out  with  sam- 
ples of  soil  oxidized  with  varying  amounts  of  hydrogen  peroxide. 
Samples  of  25  grams  of  soil  were  weighed  into  six  tared  beak- 
ers. To  each  one  was  added  50  c.  c.  of  water,  and  5 c.  c.  of  per- 
oxide was  added  to  each  of  five.  When  the  oxidizing  power  of 
the  hydrogen  peroxide  was  exhausted  one  of  the  oxidized  sam- 
ples and  the  blank  were  evaporated  to  dryness  and  dried  at 
100°  to  constant  weight.  To  each  of  the  remaining  four,  5 c.  c. 
of  hydrogen  peroxide  was  added  and  allowed  to  exhaust  itself. 
One  was  then  dried  to  constant  weight  and  the  others  treated 


12 


Wisconsin  Research  Bulletin  No.  19 


with  5 c.  c.  more  peroxide.  This  was  continued  until  oxidation 
of  each  of  samples  2,  3,  4,  5,  and  6 had  been  carried  out  with 
o,  10,  15,  20,  and  25  c.  c.  of  hydrogen  peroxide  respectively. 
Thus  five  different  stages  of  oxidation  were  obtained.  The  solu- 
bility of  the  phosphoric  acid  in  fifth  normal  nitric  acid  was  then 
determined  in  the  usual  way. 

An  increase  of  the  iron  content  of  the  extract  had  been  no- 
ticed in  samples  previously  investigated.  With  these  samples 
the  iron  in  the  fifth  normal  nitric  acid  extract  was  determined 
quantitatively  by  titrating  against  standard  potassium  perman- 
ganate after  reduction  by  a Jones  reductor.  The  results  given 
are  averages  of  duplicate  determination.  The  aluminum  oxide 
was  determined  by  subtracting  the  weight  of  phosphoric  acid 
and  ferric  oxide  from  the  weight  of  precipitate  obtained  from 
the  extract  by  addition  of  ammonium  hydroxide.  Table  Y gives 
the  results  with  three  soils.  In  two  of  the  soils  the  solubility  of 
calcium  and  manganese  was  determined. 


Table  V 


Effect  of  Different  Stages  of  Oxidation  on  the 

BILITY  OF  THE  PHOSPHORUS  OF  THE  SOIL 


SOLU- 


C.C.  30% 
H2  O2  used 

Total 

org-.  matter 
Per  cent 
of  soil 

Per  cent 
of  organic 
matter 
removed 

P2  O5  soluble 
N 5 HN03 
Per  cent 
of  soil 

Fe2  O3  soluble 
in  N/5  HNOs 
Per  cent 
of  soil 

Ala  O2  soluble 
in  N/5  HNOa 
Per  cent 
of  soil 

Soil  2x  . 

Blank 

5 

3.54 

.0 

c 

.004 

.12 

.35 

10 

• *) 
oc 

.012 

.057 

.073 

.16 

.43 

15 

. 60 

.35 

.64 

20 

.DO 

.43 

.75 

25 

.oy 

.80 

.085 

.076 

.45 

.46 

.65 

.86 

Material  Soluble  in  N/5  HNO3  Per  cent  of  Soil 


Org-.  matter 
removed 
Per  cent 
of  soil 

P2  O5 

CaO 

. 

| 

O 

w 

® 

AI2  03 

Mnl  OJ 

Soil  818 
.0 
.48 
1.52 
2.48 
3.20 
2.96 

.034 

.061 

.116 

.142 

.155 

.161 

.54 

.54 

.54 

.49 

.50 

.49 

.11 

.15 

.32 

.45 

.55 

.55 

.41 

.40 

.34 

.59 

.62 

.62 

^ y 

.22 

.25 

.31 

.21 

.24 

.24 

Soil  979 
.0 

1.08 

1.68 

2.25 

2.52 

.066 

.119 

.129 

.144 

.146 

.32 

.35 

.33 

.10 

.24 

.30 

.04 

.08 

.08 

.22 

.22 

.32 

.38 

.52 

.54 

.11 

.23 

.24 

.24 

3.20 

.143  1 

.35 

.54 

.11 

'l  his  table  shows  that  the  early  stages  of  oxidation  set  free 
much  more  phosphoric  acid  than  the  later  stages.  There 


Effect  of  Heat  and  Oxygen  on  Soil  Phosphorus  13 

.is  not  much  of  an  increase  after  one-fourth  to  one-third  of  the 
organic  matter  has  been  removed.  This  is  to  be  expected,  for  the 
•early  stages  of  oxidation  make  soluble  a large  amount  of  organic 
matter  that  is  not  really  removed  but  only  made  soluble  and  re- 
mains with  the  soil  when  the  water  is  evaporated.  When  the  soil 
is  extracted  with  nitric  acid  the  organic  matter  that  is  soluble  is 
■extracted  with  it  and  is  then  decomposed  by  bromine.  A consider- 
able part  of  the  phosphorus  may  still  be  bound  chemically  to  car- 
bon compounds  until  further  oxidation  by  bromine  in  the  nitric 
acid  extract.  The  very  last  stages  of  oxidation  set  free  no  phos- 
phoric acid  at  all.  In  soil  2x  removal  of  60  per  cent  of  the 
organic  matter  gives  just  as  much  soluble  phosphoric  acid  as  the 
removal  of  80  per  cent  does.  The  same  is  true  of  the  other  soils 
if  we  assume  that  the  highest  amount  of  organic  matter  removed 
is  80  per  cent.  It  seems  reasonable  in  the  light  of  these  facts  to 
believe  that  a removal  of  the  entire  amount  of  organic  matter 
will  not  increase  the  solubility  of  the  phosphoric  acid  above  that 
already  obtained. 

An  interesting  thing  shown  here  also  is  the  increased  solu- 
bility of  iron  and  aluminum  in  the  different  stages  of  oxidation. 
The  increase  in  the  soluble  iron  is  roughly  parallel  to  the  in- 
crease in  soluble  phosphorus  but  not  strictly  proportional  to  it. 
This  may  be  due  to  one  of  two  conditions.  The  iron  and  phos- 
phorus may  be  combined  with  the  carbon  or  they  may  be  com- 
bined with  each  other  and  not  with  carbon.  In  the  latter  case 
the  protection  from  solution  before  oxidation  must  be  by  in- 
soluble organic  matter.  The  protection  can  be  conceived  as  be- 
ing due  to  a complex  similar  to  the  one  Van  Bemeiien  believed 
to  exist  in  the  soil.  If  iron  phosphate  is  held  from  solution  in 
this  manner,  it  seems  reasonable  to  assume  that  some  of  the 
compounds  of  aluminum,  manganese  and  calcium  should  also  be 
held  from  solution  by  the  same  insoluble  complex.  This  is  seen 
to  be  the  case.  The  calcium  and  manganese  remain  constant  in 
solubility,  and  the  aluminum  varies  in  a more  irregular  man- 
ner than  the  iron  does,  but  still  increases  in  every  case.  In  soil 
818  the  amount  of  aluminum  in  the  nitric  acid  solution  is 
so  large  in  comparison  with  the  iron  that  it  was  very  difficult 
to  get  a good  basic  acetate  separation  of  the  iron  and  aluminum 
from  the  manganese.  And  in  this  soil  the  greatest  variation 
is  found  in  both  aluminum  and  manganese,  though  it  is  very 


14 


Wisconsin  Research  Bulletin  No. 


19 


small  with  the  manganese.  The  aluminum  in  soil  979  was 
determined  directly  by  the  phenyl  hydrazine  method  of  Hess 
and  Campbell  as  modified  by  Allen.2*  The  manganese  was  de- 
termined as  the  pyrophosphate.  The  results  here  seem  more 
reliable  for  these  two  metals  than  in  the  ease  of  soil  818  and 
here  there  is  an  increase  in  aluminum  closely  corresponding  to 
the  increase  of  phosphorus  and  iron.  From  these  facts  it  seems 
most  likely  that  the  iron  is  not  combined  with  the  phosphorus 
and  organic  matter  into  a single  chemical  compound.  And  it 
makes  it  appear  also  that  the  phosphorus  does  not  all  come  from 
organic  matter  but  that  some  of  it  is  present  as  phos- 
phates of  iron  and  aluminum  which  are  soluble  in  the  acid  when 
the  organic  matter  is  decomposed.  This  leads  to  the  idea  that 
iron  and  aluminum  phosphates  are  in  so  intimate  a mixture  with 
the  insoluble  organic  matter  as  to  protect  them  from  the  solvent. 

le  calcium  and  manganese  form  no  compounds  that  are  held 
m this  mixture. 

Difference  in  the  Action  of  Hydrogen  Peroxide  on  the  Sub 
pace  Soil  and  the  Subsoil 

If  the  phosphoric  acid  set  free  by  oxidation  and  heat  comes 

• r°m  °rganiC  mat,ter’.  we  shouW  not  expect  the  same  behav- 
ior with  the  subsoil  as  with  the  surface  soil.  In  the  first  place 

there  is  not  so  much  organic  matter  in  the  subsoil  as  in  the  sur- 
face soil.  Furthermore,  the  minerals  in  the  subsoil  have  never 
ieen  subjected  to  the  action  of  organic  acids  formed  by  decaying 
organic  matter  to  the  same  extent  that  the  surface  soil  has 
The  minerals  of  the  subsoil  are  therefore  likely  to  be  more  like 
the  minerals  of  the  original  rock.  If  this  is  the  case,  oxida- 
tion should  set  free  less  phosphoric  acid  from  the  subsoil  than 
from  the  surface  soil.  Heat  after  oxidation  should  have  more 
effect  on  the  subsoil  than  on  the  surface  soil.  Table  VI  shows 
results  of  experiments  along  these  lines.  Three  different  classes 
o soil  were  used:  1365  is  a virgin  clay  loam  soil;  1366  is  the 
subsoil  corresponding  to  1365;  1371  is  a sandy  loam,  virgin  soil 
and  1370  its  subsoil;  1374  is  a very  sandy  soil,  low  in  organic 
matter  and  1373  is  its  corresponding  subsoil.  The  surface  soil 

was  taken  as  the  first  eight  inches.  The  subsoil  is  eight  to 
twenty-four  inches.  s 


24  Bui.  305.  U.  S.  G.  S.  p.  95. 


Effect  of  Heat  and  Oxygen  on  Soil  Phosphorus  15 


Table  VI.  Effect  of  Heat  and  Oxidation  on  the  Soluble  Phos~ 

PHOKUS  IN  THE  SURFACE  AND  SUBSOIL 


Lab. 

No. 

Kind  of  soil 

1 

Total 
P2O5  per 
cent 
of  soil 

N /5HNO3 
soluble 
PsOs 
per  cent 
of  soil 

N/5HN03 
soluble 
P2O5 
after  oxi- 
dation, per 
cent 
of  soil 

N/5HNOs 
soluble 
P2O5 
after  oxi- 
dation and 
heat  per 
cent  of  soil 

Organic 
matter  re- 
moved, per 
cent 
of  soil 

1365 

l 

Surface  clay 
loam 

.105 

.020 

.064 

.057 

2.54 

1366 

Subsoil  clay 
loam 

.143 

.017 

.019 

.024 

.94 

1371 

Surface  sandy 
loam 

.121 

.013 

.043 

.047 

1.38 

1370 

Subsoil  sandy 
loam 

.093 

.008 

.007 

.019 

.08 

1374 

Surface  sand.. 

.069 

.018 

.033 

.035 

.60 

1372 

Subsoil  sand. . . 

.078 

.019 

.024 

.028 

.28 

The  difference  in  the  action  of  hydrogen  peroxide  on  the  phos- 
phoric acid  of  the  soil  and  subsoil  is  very  marked  with  the  clay 
and  sandy  loam  soils.  In  the  sand  the  difference  is  not  so 
marked,  and  this  is  not  unexpected  for  there  is  not  much  differ- 
ence in  the  amount  of  organic  matter.  However,  the  increase  in 
phosphoric  acid  in  the  surface  soil  is  three  times  that  of  the  sub- 
soil. Heating  gives  a larger  increase  with  the  subsoil  than  it  does 
with  the  surface  soil  after  oxidation,  though  the  increase  is  very 
much  smaller  than  it  is  with  the  surface  soil  before  oxidation. 
This  again  points  to  the  conclusion  that  the  phosphoric  acid  is 
held  by  organic  matter. 


Conclusions 

Heating  wavellite  to  200°  for  five  hours  increases  the  solubility 
of  the  phosphorus  from  4 per  cent  to  50  pe,r  cent.  Heating  it 
to  240°  for  the  same  length  of  time  increases  the  solubility  to 
100  per  cent  of  the  total  phosphorus,  as  determined  by  fusion 
with  sodium  carbonate.  Dufrenite  when  heated  to  200°  gives 
but  a slight  increase  in  solubility. 

Heating  a soil  to  100°  for  five  hours  does  not  increase  the  solu- 
bility of  phosphorus  in  fifth  normal  nitric  acid.  At  130°  a small 
increase  takes  place  and  above  this  temperature  the  solubility 
rises  rapidly  with  a rise  of  temperature,  reaching  a maximum  at 
about  200°. 

By  the  use  of  hydrogen  peroxide  about  90  per  cent  of  the 
organic  matter  of  the  soil  can  be  destroyed. 

The  solubility  of  phosphorus  is  increased  on  an  average  about 


16 


Wisconsin  Research  Bulletin  No.  19 


50  per  cent  of  the  total  phosphorus  in  clay  and  clay  loam  soils 
by  decomposing  the  organic  matter  with  hydrogen  peroxide. 
For  sandy  soils  the  increase  is  about  30  per  cent  of  the  total 
phosphorus. 

The  increase  in  the  solubility,  of  phosphorus  obtained  by  de- 
composing the  organic  matter  with  hydrogen  peroxide  is  always 
larger  than  that  obtained  by  heating  the  soil  to  200°  to  240°. 

After  the  organic  matter  has  been  destroyed  by  hydrogen  per- 
oxide there  is  no  increase  in  the  .solubility  of  phosphorus  when 
the  soil  is  heated  to  240°. 

The  excess  of  phosphorus  obtained  from  a soil  by  heating  over 
that  obtained  from  the  raw  soil  is  from  the  same  source  as  that 
obtained  by  oxidizing  with  hydrogen  peroxide. 

The  solubility  of  the  mineral  phosphates  of  the  soil  does  not 
seem  to  be  increased  by  heating  to  240°. 

The  early  stages  of  oxidation  increase  'the  solubility  of  the 
phosphorus  more  than  the  later  stages.  Much  the  greater  pare 
of  the  increase  comes  when  25  to  30  per  cent  of  the  organic  mat- 
ter has  been  destroyed.  After  60  per  cent  of  the  organic  mat- 
ter has  been  destroyed  there  is  no  further  increase  in  the  solu- 
bility of  phosphorus  on  further  oxidation. 

The  larger  increase  in  the  solubility  of  phosphorus  is  to  be 
expected  in  the  earlier  stages  of  oxidation  for  the  organic  mat- 
ter becomes  soluble  without  being  entirely  destroyed,  a further 
decomposition  being  carried  out  in  the  extract  by  bromine. 

The  solubility  of  calcium  and  manganese  is  not  increased  by 
oxidation  with  hydrogen  peroxide. 

The  solubility  of  iron  and  aluminum  in  fifth  normal  nitric 
acid  is  increased  on  oxidation  with  hydrogen  peroxide,  the  in- 
crease following  pretty  closely  the  increase  in  the  solubility  of 
the  phosphorus. 

The  increased  solubility  of  phosphorus  by  oxidation  with  hy- 
drogen peroxide  probably  comes,  in  large  part,  from  precipitated 
iron  and  aluminum  phosphates,  held  from  solution  before  the 
oxidation  as  part  of  a complex  of  insoluble  organic  matter  and 
compounds  of  iron  and  aluminum. 

Oxidation  increases  the  solubility  of  the  phosphorus  but 
slightly  in  subsoils. 

Heating  after  oxidation  has  a more  marked  effect  on  the  solu- 
bility of  the  phosphorus  in  the  subsoil  than  it  has  in  the  surface 
soil. 


(&M,  ow/Ok- 


>o 


Factors  Influencing  the  Availability  ol 
Rock  Phosphate* 


E.  TRUOG 

With  the  continually  increasing  use  of  phosphate  fertilizers, 
the  most  economical  method  of  using  them  becomes  more  and 
more  a matter  of  importance.  Numerous  field  experiments  in 
this  country,  conducted  at  the  various  experiment  stations,  and 
especially  at  Ohio,  indicate  quite  conclusively  that  when  finely 
ground  raw  rock  phosphate  is  used  in  conjunction  with  a liberal 
supply  of  organic  matter,  such  as  farm  manure  or  crop  residues, 
its  use  is  attended  with  as  large  Or  even  larger  net  profit  than 
that  attending  the  use  of  the  more  expensive  acidulated  phos- 
phates. The  increased  efficiency  of  raw  rock  phosphate  when 
supplemented  with  organic  matter  has  quite  generally  been  ex- 
plained on  the  theory  that  the  decaying  organic  matter  exerts 
a solvent  action  on  the  phosphatic  material,  and  thus  makes  it 
available  to  growing  Crops.  This  explanation  seems  reasonable, 
yet  as  a matter  of  fact  no  direct  conclusive  experimental  evi- 
dence has  ever  been  given  in  its  support.  'Attempts  have  been 
made  to  measure  this  solvent  action  by  means  of  laboratory 
experiments,  but  as  far  as  the  writer  is  aware,  this  has  never 
been  accomplished  satisfactorily.  It  was  with  a view  of  throw- 
ing more  light  on  this  subject  that  the  present  work  was  under- 
taken. 

Review  of  Previous  Work  on  This  Subject 

One  of  the  earliest  experiments  in  this  country  is  reported 
by  Lupton.* 1  Floats  were  mixed  with  cottonseed  meal  and  al- 
lowed to  ferment.  Citrate  soluble  phosphates  were  determined 


* The  author  wishes  to  express  to  Prof.  A.  R.  Whitson  his  apprecia- 
tion for  the  criticisms  and  suggestions  given  during  the  progress  of 
the  work  reported  in  this  bulletin, 

i Ala.  Exp.  Sta.  Bui.  48,  1893. 


18 


Wisconsin  Research  Bulletin  No.  20 


from  time  to  time  over  a period  of  three  months.  The  results 
as  given  a, re  irregular,  and  though  they  seem  to  indicate  a 
slight  solvent  action,  the  evidence  is  far  from  conclusive. 

A later  experiment  is  reported  by  McDowell,2  in  which  floats 
were  thoroughly  incorporated  with  mixed  cow  and  horse  manure, 
placed  in  a tight  barrel  and  allowed  to  ferment  for  a period  of 
about  thirteen  months.  Water  soluble,  citrate  soluble,  and  in- 
soluble phosphates  were  determined  at  the  beginning  and  end  ot 
the  experiment.  The  results  indicate  no  increase  in  available 
phosphates  from  the  beginning  to  the  end  of  the  experiment. 

Holdefleiss 3 composted  raw  phosphate  with  various  organic 
materials  and  inorganic  salts.  After  the  organic  materials  had 
fermented  for  eight  months,  citrate  extractions  revealed  only 
a very  slight  solvent  action  of  the  composting  materials  on  the 
raw  phosphate. 

Pfeiffer  and  Thurmann  4 made  composts  of  decaying  organic 
materials  with  raw  phosphate  and  also  with  superphosphate. 
After  these  mixtures  had  fermented  for  about  six  months,  an- 
alyses showed  that  the  raw  phosphate  had  become  but  slightly 
more  soluble  in  citrate  extraction,  while  the  solubility  of  the 
superphosphate  was  greatly  reduced  in  citrate  extraction,  and 
brought  to  almost  nothing  in  water  extraction.  Krober5  mixed 
Thomas  slag  with  sawdust  and  moist  sand.  After  fermenting 
for  three  months,  water  extractions  showed  no  increase  of  solu- 
ble phosphates. 

The  experiments  cited  are  open  to  criticism  in  that  blanks 
with  the  organic  matter  alone  were  not  carried  out  along  with 
the  others.  In  the  course  of  an  experiment  of  this  nature,  the 
available  phosphate  coming  from  the  organic  matter  itself  may 
either  increase  or  decrease.  This  increase  or  decrease  might 
then  be  sufficient  to  entirely  mask  from  chemical  measurement 
any  action  of  the  fermenting  organic  material  on  the  raw  phos- 
phates. 

Fleischer  and  Kissling,6  working  with  experiments  designed 
to  show  the  effect  of  moorland  soils  and  peaty  substances  on 
insoluble  phosphates,  found  that  they  rendered  appreciable 


2 Pa.  Exp.  Sta.  Ann.  Rpt.  ( 1907—8) , 175. 

3 Heiden,  Diingerlehre,  2,  509. 

4 Landw.  Vers.  Stat.  (1896),  343. 

'Jour.  Landw.  (1909),  57,  32. 

6 Biol.  Centr.  Agr.  Chem.  (1883),  155. 


The  Availability  of  Rock  Phosphate 


19 


amounts  of  the  phosphates  soluble  in  water  and  ammonium 
citrate.  The  solvent  action  measured  here  was  perhaps  due 
to  the  acidic  properties  of  the  substances  used.  To  a large  ex- 
tent the  nature  of  these  substances  and  their  decomposition 
products  are,  to  be  sure,  quite  different  from  that  of  farm  man- 
ure and  crop  residues,  as  used  in  ordinary  farm  practice,  and 
hence  the  results  mean  little  when  applied  to  the  problem  under 
discussion. 

Plan  of  Phosphate  Experiments 

Of  the  various  factors  influencing  the  availability  of  raw  rock 
phosphate,  the  present  investigation  includes  a study  of  the  in- 
fluence of  fermenting  cow  manure  and  June  grass;  and  the  in- 
fluence of  thorough  mixing  of  the  rock  phosphate  with  soil. 

In  the  present  consideration,  availability  of  rock  phosphate 
is  not  necessarily  taken  to  mean  its  solubility  in  weak  solvents 
only,  but  also  the  readiness  with  which  a plant  may  draw  on  it 
for  its  phosphate  supply.  To  be  sure,  the  more  soluble  a ferti- 
lizer is  in  weak  solvents,  the  more  available  it  is  to  growing 
crops.  The  converse  of  this,  however,  may  be  far  from  true. 
That  there  is  a decided  difference  between  these  two  con- 
ceptions will  be  clearly  shown  in  the  data  presented.  The 
investigations  here  undertaken  are  all  of  a laboratory  and  plant 
house  nature.  It  is  fully  recognized  that  any  data  obtained  in  this 
way  can  be  applied  to  field  conditions  only  with  the  greatest 
caution.  However,  as  already  stated,  the  field  experiments  re- 
lating to  the  problem  under  consideration  are  quite  numerous 
and  the  data  obtained  therefrom  seem  quite  conclusive,  viz, 
that  organic  matter  increases  the  efficiency  of  floats.  To  ex- 
plain why  the  field  results  are  thus,  can  only  be  accomplished 
in  the  laboratory  and  plant  house,  where  outside  disturbances 
may  be  eliminated  and  the  conditions  brought  under  control. 

THE  INFLUENCE  OF  FERMENTING  MANURE  AND 
GRASS  ON  THE  AVAILABILITY  OF  FLOATS 

Experiment  I.  Composts  of  Organic  Materials  and  Floats 

For  the  first  experiment,  jars  containing  the  mixtures  indi- 
cated, were  arranged  as  follows: 

No.  1.  2.7  kg.  sand,  25  g.  floats, 

No.  2.  2.7  kg.  sand,  25  g.  floats,  300  g.  grass. 


20 


Wisconsin  Research  Bulletin  No.  20 


No.  3.  2.7  kg.  sand, ■,  300  g.  grass. 

No.  4.  2.7  kg.  sand,  25  g.  floats,  300  g.  manure. 

No.  5.  2.7  kg.  sand,  : , 300  g.  manure. 

Materials  Used  The  ja,rs  were  1-gallon,  glazed,  earthenware 
vessels,  each  provided  with  a hole  in  the  bottom.  The  sand  was 
ground  quartzite  from  Wausau,  Wis.  analyzing  97.9  per  cent 
silica.  The  floats  consisted  of  a high  grade  of  finely  ground  rock 
phosphate,  analyzing  34  per  cent  phosphoric  anhydride.  The 
grass  was  finely  chopped',  fresh,  green,  June  grass.  The  manure 
was  fresh  cow  manure  without  litter. 

The  contents  of  each  jar  were  thoroughly  mixed.  The  jars 
were  placed  in  the  green  house,  maintained  at  optimum  water 
content  or  nearly  so,  and  stirred  occasionally.  Active  fermen- 
tation soon  appeared  to  take  place  in  all  the  jars  containing  or- 
ganic matter. 

This  work  was  started  about  August  1.  On  the  next  December 
10,  the  material  in  each  jar  was  extracted  with  water,  and 
several  weeks  later  extractions  were  made  with  0.2  per  cent  citric 
acid  and  1 per  cent  sodium  hydroxide  solution.  The  extrac- 
tions were  made  as  follows : 

Water  Extraction  Water  was  applied  to  the  jars  until  the 
teachings  passing  through  the  holes  in  the  bottoms  amounted 
to  two  liters.  These  solutions  were  filtered  till  clear  from  all 
suspended  material  and  then  analyzed  for  phosphoric  acid. 

Citric  Acid  Extraction  Duplicate  1/20  portions,  making 
about  150  g.  were  weighed  out  from  each  jar,  placed  in  flasks 
and  extracted  with  300  c.  c.  of  0.2  per  cent  cit,ric  acid.  The 
flasks  were  shaken  occasionally  for  several  hours  and  then  allow- 
ed to  stand  24  hours,  when  25  c.  c.  portions  were  drawn  off, 
filtered  and  analyzed  for  phosphoric  acid.  After  standing  eight 
days  with  frequent  shaking,  portions  were  again  removed  for 
analysis. 

Sodium  Hydroxide  Extraction  For  this,  1/50  portions  were 
taken  from  each  jar,  placed  in  flasks  and  extracted  with  1 per 
cent  sodium  hydroxide  solution,  according  to  the  method  of 
Stoddart.7  The  resulting  solutions  were  theu  analyzed,  as  in 
the  former  extractions. 


T Wis.  Exp.  Sta.  Res.  Bu\,  2,  1909,  p.  §3. 


The  Availability  of  Rock  Phosphate 


21 


Method  for  Determining  Phosphoric  Acid  Except  where 
otherwise  stated  the  analyses  of  the  various  extracts  for  phos- 
phoric acid  reported  in  this  work  have  been  made  colorimetric- 
ally.  The  method  used  and  given  herewith  is  the  writer  ’s  mo- 
dification of  the  method  given  in  U.  S.  Bureau  of  Soils  Bulletin 
31,  page  45. 

A measured  quantity  of  the  clear  solution  to  be  analyzed  is 
evaporated  in  a casserole  to  a volume  of  about  20  c.  c.  The 
solution  is  made  alkaline  with  sodium  hydroxide  and  just  a 
slight  excess  added.  The  casserole  is  covered  and  bromine  ad- 
ded from  a burette  through  the  lip  of  the  casserole  in  small 
quantities  from  time  to  time,  heating  on  the  water  bath.  After 
treating  in  his  way  fop  about  one  hour,  dilute  nitric  acid  is 
added  till  the  bromine  is  all  driven  off,  and  then  the  solution 
evaporated  to  dryness.  If  this  process  has  been  properly  car- 
ried out,  the  organic  matter  will  be  entirely  destroyed  and  the 
residue  perfectly  colorless.  The  residue  is  then  dehydrated  at 
110 ? C.  for  two  hours.  To  this  residue  5 c.  c.  nitric  acid,  sp.  gr. 
1.07,  are  added  with  a little  water.  The  solution  is  filtered,  and 
the  casserole  and  filter  washed  with  water  till  the  filtrate  meas- 
ures about  40  c.  c.  This  leaves  most  of  the  silica  adhering  to 
the  bottom  of  the  casserole,  the  filter  catching  any  that  may 
wash  out.  Prom  here  on  the  reagents  are  used  and  the  color 
developed  as  described  in  the  bulletin  referred  to. 

It  is  essential  to  use  silica-free  water  and  to  keep  reagents 
in  paraffin  lined  bottles.  In  making  comparisons  the  standard 
and  unknown  should  be  at  the  same  temperature,  and  the  dilu- 
tions should/  be  such  that  approximately  equal  volumes  of  the 
standard  and  unknown  are  compared. 

By  this  method  of  treatment  with  bromine,  all  organic  matter 
is  destroyed  and  hence  cannot  interfere  with  the  subsequent  de- 
velopment of  color.  The  phosphorus  in  the  soluble  organic 
material  is  thus  determined.  This  method  has  been  used  with 
success  in  this  laboratopy  for  several  years,  during  which  time 
a large  number  of  determinations  have  been  made-.  Its  value 
lies  in  that  small  amounts  of  phosphates  can  be  detected  and 
determined  without  laboriously  evaporating  down  large  volumes. 
Then  again,  in  experiments  of  this  kind,  the  total  volume  of 
solution  at  hand1  might,  not  give  enough  material  to  determine 
gravimetrically.  On  several  occasions  we  have  checked  the 
method  with  the  gravimetric  method  and  usually  obtained  re- 


22 


Wisconsin  Research  Bulletin  No.  20 


markable  concordance.  In  cases  where  the  quantitative  relation 
between  the  two  varied  somewhat,  the  comparative  relation  be- 
tween the  members  of  a set  determined  colorimetrically  still  had 
very  closely  the  same  relation  as  when  the  set  was  determined 
gravimetrically.  For  rapid  comparative  work  the  method  is 
thus  reliable  and  can  be  depended  upon  to  show  small  differ- 
ences which,  if  of  any  importance,  may  then  be  checked  up  by 
gravimetric  or  volumetric  determinations. 

Table  I gives  the  results  of  the  analyses  of  the  extracts  se- 
cured in  the  manner  already  described  with  the  different  sol- 
vents. 


Table  I Parts  of  Phosphoric  Anhydride  per  Million  Parts  of 
the  Extracting  Solutions 


Jar 

Treatment 

Solvent  Used 

Ps  Os 
by  water 

Pj  Os  by 
0.2%  citric 
acid  24  hrs. 

P 2 Os  by 
0.2%  citric 
acid  8 days 

Ps  Os  by 
1%  NaOH 

1 

Quartz  and  floats 

1.5 

116.0 

149.0 

7.5 

O 

Quartz,  floats  and 

grass 

88.0 

68.5 

99.0 

11.0 

3 

Quartz  and  grass 

88.0 

26.0 

32.0 

8.7 

4 

Quartz,  floats  and 

manure 

71.0 

88.0 

118.0 

12.5 

5 

Quartz  and  manure... 

67.0 

66.5 

57.0 

9.3 

From  this  table  it  is  quite  clear  that  neither  the  fermenting 
grass  nor  the  manure  had  any  material  effect  on  the  solubility 
of  the  floats  as  measured  by  the  extracting  water.  The  con- 
tention is  not  made,  however,  that  there  has  been  no  solvent 
action,  but  simply  that  this  extraction  measures  none.  The  dif- 
ference between  Nos.  (4)  and  (5)  indicates  nothing  since  No. 
(1)  must  be  added  to  No.  (5)  to  make  the  two  comparable. 
When  thisi  is  done,  the  difference  is  only  about  3.5  per  cent, 
easily  within  the  limit  of  error  of  the  chemical  work. 

The  extraction  with  0.2  per  cent  citric  acid  also  fails  to  meas- 
ure any  solvent  action  that  may  have  taken  place.  As  a matter  of 
fact,  when  we  compare  No.  (1)  with  Nos,  (2)  and  (4),  it  becomes 
quite  clear  that  the  fermenting  organic  matter  has  rendered  the 
floats  less  available,  when  availability  is  measured  by  a solvent 
such  as  .2  per  cent  citric  acid.  If  we  consider  the  24  hour  ex- 
traction, and  subtract  66.5  from  88,  it  leaves  21.5  parts.  This 
we  may  take  as  the  citric  acid  soluble  phosphates  coming  from 
the  floats  in  No.  (4).  When  the  figure  116  in  No.  (1)  is  now 


The  Availability  of  Rock  Phosphate 


23 


compared  with  21.5,  it  indicates  by  this  method  of  reasoning 
that  the  manure  has  decreased  the  solubility  of  the  floats  in  0.2 
per  cent  citric  acid  by  more  than  five  times.  The  same  calcula- 
tion shows  that  the  grass  has  decreased  it  about,  three  times. 
Nevertheless  no  one  would  hardly  dare  to  maintain  that  the  mix- 
ing of  floats  with  farm  manure,  as  is  done  in  ordinary  farm 
practice,  results  in  the  floats  becoming  less  available  to  the  grow- 
ing crop  due  to  the  presence  of  the  manure.  This  point  has 
already  been  referred  to  and  will  be  again  taken  up  in  a further 
discussion.  That  availability  as  measured  by  weak  solvents 
may  be  entirely  different  from  that  as  measured  by  growing 
crops  seems  quite  evident. 

The  extraction  with  sodium  hydroxide  was  made,  thinking 
that  perhaps,  due  to  the  presence  of  some  iron  in  the  quartz 
and  floats,  the  phosphates  might  go  over  into  iron  phosphates 
as  soon  as  made  soluble.  This  being  the  case,  the  sodium  hy- 
droxide extraction,  which  readily  dissolves  iron  phosphates, 
should  show  that  No.  (2)  is  larger  than  the  sum  of  No.  (1)  and 
No.  (3)  and  the  same  for  the  other  set.  The  figures  do  not  show 
this  and  hence  the  contention  is  untenable  as  far  as  this  method 
of  extraction  is  concerned. 

General  Inferences  From  Experiment  I 

The  data  in  Table  I plainly  show  that  the  methods  used  in 
Experiment  I failed  to  measure  any  solvent  action  of  the  fer- 
menting organic  matter  on  the  floats.  Why  should  this  be  the 
case?  Can  it  be  that  there  was  no  action?  The  fact  that  the 
organic  matter  made  the  floats  less  available  in  0.2  per  cent 
citric  acid  solution  indicates  that  the  floats  were  acted  upon  in 
some  way  by  the  organic  matter,  either  chemically,  physically, 
or  both.  It  seemed  quite  .reasonable  to  suppose  that  the  organic 
matter  had  made  the  floats  soluble  to  a certain  extent,  and  that 
this  soluble  portion  was  immediately  absorbed  and  held  .physic- 
ally and  chemically  by  the  organic  matter  in  such  a way  that 
the  citric  acid  solution  would  not  extract  it.  The  results  of 
Pfeiffer  and  Thurmann,  as  already  given,  touch  directly  on  this 
question.  Their  investigations  show  that  the  composting  of  or- 
ganic matter  with  acid  phosphate  greatly  reduces  the  solubility 
of  the  phosphate  in  water  and  citrate  extractions.  These  re- 
sults together  with  the  fact  that  phosphates  more  than  any  other 


24 


Wisconsin  Research  Bulletin  No. 


20 


•silts,  are  known  to  be  absorbed  and  held  by  organic  matter  with 
such  .retentiveness  that  it  is  difficult  to  .extract  them  with  weak 
solvents,  greatly  strengthened  the  foregoing  supposition. 

Bacteria  and  other  soil  organisms  undoubtedly  use  up  a por- 
tion of  the  soluble  phosphates  in  their  own  life  processes.  In 
this  case  the  portion  used  would  be  locked  up  in  the  bodies  of 
he  organisms  and  would  not  be  measured  by  a weak  solvent.  As 
to  the  rapidity  with  which  this  process  takes  place  the  reader 
is  referred  to  further  discussion  on  page  28. 

If  the  supposition  that  the  soluble  phosphates  are  held  phy- 
sically is  correct,  then  a method  which  will  destroy  the  remain- 
mg  organic  matter  and  release  the  phosphates  just  before  the 
extraction  is  made,  should  prove  successful  in  measuring  the 
solvent  action.  Also  if,  as  Pfeiffer  and  Thurmann  have  shown, 
the  mixing  of  acid  phosphate  with  organic  matter  renders  the 
phosphate  less  easily  extractable  by  a weak  solvent,  then  we  will 
have  additional  evidence  in  favor  of  the  contention  in  the  pre- 
ceding paragraph.  For  the  purpose  of  testing  these  possibil- 
ities the  following  experiment  was  conducted. 


Experiment  II.  Composts  of  Manure  and  Floats,  and  Manure 
and  Acid  Phosphate 

For  the  investigations  in  this  experiment  the  following  jars 
were  arranged,  observing  the  utmost  care  in  the  selection  of 
materials,  the  mixing  and  arrangement  of  the  same  and  the  sub- 
sequent care  during  the  period  allowed  for  fermentation. 

No.  1.  6 kg.  quartz  

No.  2.  6 kg.  quartz  25  g.  floats  

No.  3.  6 kg.  quartz  loo  g.  manure. 

No.  4.  6 kg.  quartz  25  g.  floats  100  g.  manure. 

No,  5.  6 kg.  quartz  10  g.  acid  phosphate  - 

No.  6.  6 kg.  quartz  10  g.  acid  phosphate  100  g.  manure 

Materials  Used  The  jars  were  1 -gallon  glazed  earthenware 
vessels  without  holes.  The  quartz  was  a natural  sand  from 
Ottawa,  111.,  analyzing  99.13  per  cent  silica.  The  floats  consisted 
of  a high  grade  of  finely  ground  rock  phosphate  analvzing  35  3 
per  cent  phosphoric  anhydride.  The  acid  phosphate  was  acidul- 
ated rock,  containing  14  per  cent  available  and  15  per  cent  total 
phosphoric  anhydride.  The  manure  consisted  of  finely  ground 
air  dried  cow  manure  without  litter. 


The  Availability  of  Rock  Phosphate 


25 


The  whole  series  of  jars  was  arranged  in  duplicate,  making 
twelve  in  all.  The  experiment  was  thus  duplicated  from  the 
starting  of  the  work  with  the  jars  to  the  end  of  the  chemical 
work.  The  phosphatic  materials  were  thoroughly  mixed  with 
the  quartz  before  adding  the  manure.  The  manure  being  dry 
and  finely  ground  was  easily,  thoroughly,  and  uniformly  mixed. 
In  order  to  insure  uniform  bacterial  activity,  each  jar  received 
a little  water  extract  from  a rich  soil.  To  each  jar  825  c.  c.  of 
water  were  added.  The  jars  were  then  weighed.  This  gave  a 
standard  weight  for  each  jar  at  which  the  per  cent  of  moisture 
for  contents  of  all  was  practically  the  same..  The  jars  were 
kept  in  the  plant  house  and  watered  to  standard  weight  once  a 
week,  stirring  after  each  watering. 

This  method  of  procedure  is  preferable,  not  only  in  that  it 
makes  it  possible  to  keep  the  material  in  the  different  jars  at 
the  same  moisture  content,  and  hence  more  nearly  under  the 
same  conditions,  but  also  in  that  it  eliminates  the  necessity  of 
making  moisture  determinations  whenever  samples  are  taken  for 
analysis.  Whenever  samples  were  taken  in  the  following  work, 
the  jars  were  first,  watered  up  to  standard  weight  and  the  con- 
tents thoroughly  mixed.  A given  weight  of  material  from  any 
jar  was  then  strictly  comparable  to  the  same  weight  from  any 
other  jar.  The  amount  of  material  taken  from  any  jar  was 
always  recorded  in  order  that  the  jar  might  be  given  the  correct 
new  standard  weight,  at  which  the  moisture  per  cent  of  contents 
was  unchanged  from  former  standard  weight.  This  set  of  jars 
was  started  February  26.  On  the  next  June  11,  samples  were 
taken  for  the  following  extractions : 

Water  Extractions  Fifty  gram  samples  were  put  into  600 
c.  c.  Erlenmejyer  flasks  and  250  c.  c.  of  water  added.  The 
flasks  were  shaken  several  times  and  then  allowed  to  stand  24 
hours,  when  portions  of  the  solutions  were  filtered  and  analyzed 
for  phosphates. 

Hydrogen  Peroxide  Oxidation  and  Subsequent  Extraction 
In  order  to  investigate  the  possibility  of  manure  absorbing 
and  holding  physically  any  phosphates  that  had  been  made  solu- 
ble as  suggested  on  page  23,  the  following  method  of  oxidation 
and  extraction  was  used.  Samples  of  25  g.  each  were  put  into  300 
c.  c.  Erlenmeyer  flasks.  From  the  jars  numbered  (3)  contain- 


26 


Wisconsin  Research  Bulletin  No.  20 


ing  quartz  and  manure,  a double  set  of  samples  was  taken  To 
one  of  these  sets  now  called  (3a),  .09  g.  of  floats  was  added. 
Hus  » the  amount  of  floats  carried  by  a 25  g.  sample  from  jar 
(4),  which  received  both  manure  and  floats  in  the  beginning, 
dins  set  (3a)  was  taken  as  a check  against  the  possibility  that 
the  oxidation  of  the  organic  matter  with  the  hydrogen  peroxide 
might  m itself  render  a part  of  the  floats  soluble.  A little  water 
was  added  to  each  flask  and  then  3 per  cent  hydrogen  peroxide 
m portions  of  10  c.  c.  The  peroxide  was  a dilution  of  Merck’s 
pure  30  per  cent  solution.  The  flasks  were  set  on  the  water  bath 
and  gently  warmed.  After  adding  about  30  c.  c.  of  the  per- 
oxide, practically  all  of  the  organic  matter  was  oxidized  and  the 
solutions  were  perfectly  colorless.  The  solutions  obtained  were 
neutral  to  litmus,  indicating  that  the  bases  and  acids  liberated, 
just  about  neutralized  each  other.  The  solutions  were  filtered 
and  the  residue  thoroughly  washed  with  water  to  remove  all 
soluble  material.  The  filtrates  were  made  up  to  definite  volume 
and  portions  taken  for  analyses.  The  water-insoluble  residues 
from  (3)  and  (3a)  were  further  treated  as  follows:  The  filter 
papers  containing  part  of  the  material  were  returned  to  the 
respective  flasks.  These  residues  were  then  treated  with  0.02 
per  cent  citric  acid  solution.  The  extractions  covered  a period 
of  one  hour  during  which  time  the  flasks  were  frequently  shaken. 
Filtered  portions  were  then  analyzed  for  phosphates. 

Table  II  gives  the  results  of  the  analyses  of  the  extracts  se- 
cured by  the  methods  just,  described. 


Table  II  Parts  of  Phosphoric  Anhydride  per  Million  Parts 
op  Extracting  Solutions 


| 

Before 

oxidation 

After  H3O2  oxidation 

.Tar 

Treatment 

P2O5  by 
ater. 

Ps05  by 
water 

PsOs  by  0.02% 
| citric  acid 

1 

2. . . 

Q 

Quartz 

Quartz  and  floats.... 

0.4 

1.3 

0.3, 

1.2 

O 

4  

5  

Quartz, , manure 

Quartz,  floats,  manure  . 

Quartz,  acid  phosphate  

14.0 

16.0 

20.0 

20.0 

18.6  „ 

6 

Quartz,  acid  phosphate,  manure.. 
Same  as  3 with  floats  added  just 
before  oxidation 

9.0 

23.0 

8.5 

3a.... 

28.0 

20.7 

18.2  ' 

Water  Extraction  before  Oxidation  In  this  table  it  is 
to  be  noticed  that  the  results  with  water  extraction  are 


The  Availability  of  Pock  Phosphate 


27 


similar  to  those  in  Table  I.  When  the  sum  of  numbers  (2)  and 
(3)  is  compared  with  number  (4),  the  difference  is  again  within 
the  limit  of  error  and  hence  no  definite  solvent  action  has  been 
measured.  In  the  case  of  the  acid  phosphate,  the  sum  of  num- 
bers (3)  and  (5)  just  equals  number  (6).  This  indicates  that 
soluble  phosphates  as  found  in  acid  phosphate  may  be  mixed 
and  left  with  decaying  manure,  in  such  proportions  as  used 
here,  for  several  months,  after  which  water  will  again  readily 
extract  the  greater  part  of  the  phosphates. 

It  is  important  to  note,  however,  that  the  conditions  which 
influence  chemical  fixation  of  phosphates  were  quite  different 
in  the  jars  containing  acid  phosphate  and  manure  from  those 
containing  floats  and  manure.  The  10  g.  of  acid  phosphate 
used  with  the  manure  were  sufficient  to  maintain  the  contents 
of  the  jars  decidedly  acid  to  litmus  throughout  the  experiment. 
The  contents  of  the  jars  containing  the  floats  and  manure  devel- 
oped a slightly  alkaline  reaction  to  litmus,  as  did  also  a boiled 
extract  of  the  same  to  phenolphthalein.  Thus  in  the  case  of  the 
acid  phosphate,  there  was  little  chance  for  chemical  fixation 
of  phosphates,  since  an  excess  of  acid  was  always  present.  In 
the  case  of  the  rock  phosphate,  it  is  quite  possible  that  the  alka- 
line manure  medium  served  to  fix  chemically  part  of  the  phos- 
phates that  may  have  been  made  soluble.  It  is  thus  evident 
that  the  two  cases  cannot  be  compared  in  every  way  as  was 
originally  planned.  However,  the  results  do  seem  to  indicate 
that  the  amount  of  water-extractable  phosphates  is  affected  but 
little  by  physical  fixation  under  conditions  as  obtained  with  the 
mixtures  of  acid  phosphate  and  manure  used  in  the  present 
experiment. 

The  results  with  acid  phosphate  may  appear  to  be  contradic- 
tory to  what  Pfeiffer  and  Thurmann  found.  These  investiga- 
tors, however,  used  a considerably  larger  proportion  of  organic 
matter  to  acid  phosphate  than  was  used  in  the  present  work, 
which  seems  to  explain  the  difference ; for  the  larger  the  propor- 
tion of  organic  material,  the  more  likely  is  the  media  to  become 
alkaline  and  hence  bring  about  chemical  fixation  and  possibly 
aid  physical  fixation. 

Fixation  of  phosphates  due  to  bacterial  activity  would  proba- 
bly also  be  larger  in  the  alkaline  medium  than  in  the  acid  med- 
ium. 


28  Wisconsin  Research  Bulletin  No.  20 

Water  Extraction  after  Oxidation  The  figures  in  the  sec- 
ond column  show  that  the  extraction  with  water,  after  the  hy- 
drogen peroxide  oxidation,  has  also  failed  to  measure  any  sol- 
vent action  of  the  manure  on  the  floats. 

Citric  Acid  Extractions  after  Oxidation  As  was  indicated 
with  the  acid  phosphate,  the  failure  to  measure  any  solvent 
action,  by  water  extraction  does  not  now  seem  to  be  due  to 
physical  absorption  and  retention,  but  .rather  to  the  possibility 
that  the  phosphates  as  soon  as  made  soluble,  are  again  precipi- 
tated by  the  alkaline  manure  medium  or  partially  used  up  by 
bacterial  activity.  The  action  of  the  fermenting  manure  on 
the  floats  would  thus  result  in  a splitting  up  of  the  particles 
of  floats  into  very  much  smaller  particles — molecules.  If  this 
is  the  case,  then  the  particles  of  rock  phosphate  left  after  oxid- 
ation and  water  extraction  in  the  residue  of  No.  (4),  where 
manure  fermented  in  the  presence  of  floats,  should  he  more 
finely  divided  on  the  whole  than  those  in  No.  (3),  where  the 
floats  were  not  added  until  the  oxidation  process.  This  being 
the  case,  a weak  solvent  like  0.02  pe,r  cent  citric  acid  should1 
extract  more  phosphates  from  the  former,  where  the  material 
is  more  finely  divided,  and  part  of  which  has  been  freshly 
precipitated.  The  figures  in  the  last  column  of  Table  II  give  the 
results  of  this  extraction.  The  difference,  while  being  in  favor 
of  No.  (4),  indicating  a solvent  action  of  the  manure,  is  easily 
within  the  limit  of  error.  The  work  was  repeated,  when  the 
results  also  favored  No.  (4)  but  were  again  within  the  limit  of 
error. 

Had  any  considerable  portion  of  phosphate  become  locked 
up  in  bacterial  cells,  then  the  hydrogen  peroxide  oxidation 
would  have  released  this  portion  again  and  left  it  in  a condi- 
tion more  soluble  in  0.2  per  cent  citric  acid,  than  the  original 
rock  phosphate.  The  .results  in  the  last  column  of  Table  II 
bear  directly  on  this  point.  While  these  results  do  not  contra- 
dict the  possibility  that  phosphates  have  been  locked  up  in  bac- 
terial cells,  yet,  they  do  indicate  that  under  conditions  as  ob- 
tained in  this  experiment,  the  locking  up  of  phosphates  in  this 
way  is  a comparatively  slow  process. 

Citric  Acid  Extractions  Under  Varying  Conditions  Since  the 
data  in  Table  I show  that  the  manure  has  made  the  floats  less 
soluble  in  0.2  per  cent  citric  acid  solution,  it  seemed  desirable  to 
confirm  these  data  with  the  present  set  of  jars.  It  also  seemed  de- 


The  Availability  of  Rock  Phosphate 


29 


sirable  to  investigate  how  the  results  might  he  influenced  by 
varying  the  length  of  period  of  extraction  and  further  by 
varying  the  ratio  of  solvent  to  weight  of  material  extracted. 
Accordingly,  July  26  the  jars  were  watered  to  standard  weight 
and  three  sets  of  samples  taken  and  extracted  according  to  the 
following  scheme : 

Set;  I.  100  g.  sample,  with  100  c.  c.  acid.  Ratio,  1 :1 
Set  II.  100  g.  sample,  with  300  c.  c.  acid.  Ratio,  1 :3 
Set  III.  25g.  sample,  with  250  c.  c.  acid.  Ratio,  1 :10 
From  jars  numbered  (3),  containing  manure  and  quartz, 
twice  as  many  samples  were  taken  as  from  the  others.  To  one- 
half  of  these  now  called  (3a)  floats  were  added  in  an  amount 
equivalent  to  that  carried  by  samples  from  jars  numbered  (4). 

The  samples  were  placed  in  Erlenmeyer  flasks,  the  0.2  per 
cent  citric  acid  added  and  then  the  flasks  were  shaken  alter- 
nately for  one-half  hour,  then  let  stand  one-half  hour,  when 
small  portions  of  about  20  c.  c.  were  filtered  off  and  10  c.  c.  por- 
tions taken  for  analysis.  Any  unused  solution  with  filter  papers 
and  contents  was  always  returned  to  the  respective  flasks.  At 
the  end  of  six  and  twenty-four  hour  periods,  portions  were  again 
removed  for  analysis,  the  flasks  being  shaken  occasionally  dur- 
ing the  meantime.  Table  III  gives  the  results  of  these  extrac- 
tions. 


Table  III  Parts  of  Phosphoric  Anhydride  per  Million  of  Solu- 
tion as  Extracted  by  0.2  per  cent  Citric  Acid  Under  Differ- 
ent Conditions  of  Extraction. 


No. 

Treatment  of  quartz 

100  g.  sample 
100  c.  c.  acid 
Ratio,  1.1 

100  g.  sample 
300  c.  c.  acid 
Ratio,  1:3 

25  g sample 
250  c.  c.  acid 
Ratio,  1:10 

1 hr. 

6 hr. 

24  hr. 

1 hr. 

6 hr. 

24  hr. 

1 hr. 

6 hr. 

24  hr. 

2 

Floats 

85 

98 

122 

59 

92 

116 

34 

55 

86 

B 

Manure 

122 

130 

132 

46 

54 

53 

18 

18 

19 

4 

Manure  and  floats 

138 

144 

158 

69 

96 

110 

40 

59 

86 

3a 

(a) 

128 

132 

148 

66 

94 

106 

43 

64 

94 

4a 

(b) 

16 

14 

20  j 

23 

42 

57 

22 

41 

67 

(a)  Same  as  (3),  with  floats  added  just  before  wfidatiiow. 

(b)  Gives  remainders  when  results  under  (B)  are  subtracted  from  (4)  and  repre- 
sents phosphates  coming1  from  floats  in  (4). 


It  is  important  to  note  that  in  this  Table  the  order  of  results 
has  been  changed  considerably  by  altering  the  conditions  of  ex- 
traction. Where  the  ratio  of  solid  to  solvent  was  1 :3  and  the 


30 


Wisconsin  Research  Bulletin  No.  20 


period  of  extraction  24  hours,  which  conditions  are  much  similar 
t°  those  that  prevailed  in  the  citric  acid  extraction  reported  in 
Table  I,  the  results  are  also  similar  to  those  reported  in  that 
table,  viz:  that  more  phosphates  were  extracted  from  the  floats 
alone  than  from  the  mixture  of  floats  and  manure  which  had 
been  undergoing  fermentation  in  intimate  contact  for  five 
months.  There  does  not  seem  to  be  any  good  reason  why  the 
extractions  should  not  have  obtained  just  as  much  soluble  phos- 
phates from  the  manure  itself  in  No.  (4)  as  in  No.  (3).  If  we 
assume  that  the  soluble  phosphates  extracted  from  the  manure 
in  the  two  cases  were  equal,  then  the  total  extraction  of  No. 
(4)  as  given  in  the  table  minus  No.  (3)  gives  the  quantity  of 
phosphates  coming  from  the  floats  in  No.  (4).  The  figures  fol- 
lowing (4a)  give  these  quantities  after  making  the  subtractions. 
On  comparison  we  find  that  these  quantities  a,re  always  much 
less  than  those  following  No.  (2).  This  is  again  similar  to  the 
results  in  Table  I,  and  indicates  that  the  mixing  of  floats  with 
manure  makes  the  floats  less  soluble  in  weak  citric  acid  solution. 
It  is  to  be  noted,  as  might  be  expected,  that  this  difference  be- 
tween Nos.  (2)  and  (4a)  becomes  less  as  the  ratio  of  solvent  in- 
creases. 

The  figures  following  (3a)  are  somewhat  irregular,  but  as  a 
general  average  thejy  follow  No.  (4)  quite  closely.  These  fig- 
ures under  (3a)  will  be  considered  again  in  a further  discus- 
sion. 

The  data  in  Table  III  become  more  easily  interpreted  when 
the  use  of  curves  is  resorted  to.  The  data  for  Nos.  (2),  (3),  (4), 
and  (4a)  have  been  plotted  in  Figures  1,  2,  and  3,  using 
parts  per  million  as  ordinates  and  time  in  hours  as  abscissae.8 

The  curves  for  No.  (3)  show  that  all  the  soluble  material  in 
the  manure  went  into  solution  during  the  first  one  or  two  hours 
of  extraction,  the  curves  becoming  practically  horizontal  there- 
after. The  curves  for  No.  (4)  rise  quickly  at  first  due  to  the 
soluble  phosphates  present  in  the  manure  itself.  At  the  end  of 
one  or  two  hours,  soluble  phosphates  ceased  to  come  from  the 
manure  and  then  the  rise,  being  due  solely  to  phosphates  com- 
ing into  solution  from  the  floats,  becomes  much  slower.  As  a 
matter  of  fact,  the  rise  is  then  slower  in  all  other  cases  than  in 


s Drawings  fo-r  Figures  1,  2,  3,  and  4 were  made  by  E.  R.  Finner  of 
the  Dept,  of  Soils. 


The  Availability  Of  Rock  Phosphate  31 

No.  (2),  where  no  manure  was  in  contact  with  the  floats.  Where 
the  ratio  was  1 :3,  the  manure  has  lessened  the  solubility  of  the 
floats  so  much  that  No.  (2)  is  ahead  of  No.  (4)  at  the  end  of 
24  hours,  and  where  the  ratio  was  1:10  No.  (2)  has  just  caught 
up  with  No.  (4).  When  the  curves  for  No.  (4a)  are  compared 
with  those  for  No.  (2)  the  comparisons  serve  to  bring  out  in 
a striking  way  how  the  presence  of  the  manure  in  contact  with 
the  floats  has  lessened  the  solubility  of  the  floats  in  the  citric 
acid  extractions. 

a 

As  already  stated,  the  figures  under  No.  (3a)  are  somewhat 
irregular  and  unsatisfactory.  If  No.  (3a)  is  accepted  as  a per- 
fect blank  on  No.  (4),  then  the  two  extractions  where  the  ratios 
were  1 :1  and  1 :3  indicate  a slight  solvent  action  of  the  manure 
on  the  floats.  The  other  extraction,  however,  indicates  the  op- 
posite. In  providing  for  samples  No.  (3a)  it  was  necessary  to 
mix  the  floats  with  wet  manure  and  quartz.  To  get  thorough 
and  uniform  mixing  in  a case  like  this  is  a difficult  problem.  It 
seems  that  the  thorough  shaking  after  adding  the  solvent  should 
have  resulted  in  getting  efficient  mixing. 

In  order  to  check  up  this  part  of  the  work,  another  set  of 
samples  was  taken,  observing  the  utmost  care  tc  secure  efficient 
mixing.  Samples  of  100  g.  were  extracted  with  100  c.  c.  of  the 
acid.  In  order  to  secure  uniform  shaking,  the  flasks  containing 
the  mixtures  were  shaken  in  a mechanical  shaker  for  one  half 
hour,  after  which  portions  were  filtered  off  and  analyzed.  The 
determinations  were  made  volume trically  by  solution  of  the 
ammonium-phosphomolybdate  precipitate  in  standard  alkali  and 
titration  of  excess  with  acid.  This  method  was  deemed  prefera- 
ble to  the  gravimetric  method,  since  it  is  better  adapted  to  show 
small  differences.  The  results  of  this  work  are  given  in  Table 
IV. 


Table  IV  Pabts  of  Phosphoric  Anhydride  per  Million  of  Solu- 
tion, as  Extracted  from  Different  Mixtures 


No. 

Treatment  of  Quartz 

Parts  P2O5  by 
.2%  citric  acid 

2 

Floats 

92.0 

169.0 
193.5 

193.0 

3 

Manure 

4 

Manure  and  floats 

3a.. . . 

(a) 

(a)  Same  as  No.  3 except  that  floats  were  added  just  before  extraction  in  an 
amount  equal  to  that  carried  by  No.  4. 


32 


Wisconsin  Research  Bulletin  No.  20 


eg  > 

;*> 

• 

TT 

l! 

• 

! 

• 

r 

i' 

• 

| 

• 

• 

| 

• 

fi 

ii 

• 

i 

i 

1 1 

• 

i 

i 

1 1 

» 

M 

1! 

i 

i 

~t — 1 

1 

~i — : — 

1 

i | 
1 
j 

r 

ii 

\ 

ft 

| \ 

ti 
i i 

j i_ 

i i 

n 

u. 

•\ 

n 

i 

*0 

•\ 

r 

i 

• \ 

r 

# X 

w 

\ 

\ 

V 

\ 

o 

> 

\ 

V 

§ I % S $ * § * 

uo/jn/oS'  u/ 

uouhuj  joJ  SQzy  SJJOcJ 


Figure  2.  Rate  of  solution  of  phosphates  similarly  as  in  Figure  1,  when  the  ratio  of  solid  to  solvent  was  1:3. 


The  Availability  of  Pock  Phosphate 


33 


uo/jn/os  ut 

UO////LU  ssd  so  ?c/  sjsOcJ 


Figure  3.  Rate  of  solution  of  phosphates,  similarly  as  in  Figure  1,  when  the  ratio  of  solid  to  solvent  was 


^ Wisconsin  Research  Bulletin  No.  20 

* rom  this  table  it  is  evident  that  little  solvent  action  has  been 
measured,  since  No.  (4)  and  No.  (3a)  are  practically  the  same 
the  other  results  are  exactly  in  the  same  order  as  in  Table  III. 
The  vigorous  shaking  has  resulted  in  bringing  much  more  phos- 
phates into  solution  in  a short  time  than  was  obtained  by  the 
previous  extractions. 

General  Consideration  of  Experiments  I and  II 

From  the  data  which  have  been  presented  so  far  it  is  quite 
evident  that  the  solution  of  the  problem  under  consideration  is 
attended  with  many  difficulties.  Why  the  presence  of  the  man- 
ure m contact  with  the  floats  should  so  greatly  reduce  the  solu- 
bility of  the  floats  m dilute  citric  acid  solution  is  not  altogether 
clear.  That  it  is  not  due  to  the  locking  up  of  the  phosphates 
m bacterial  cells  is  evident  from  the  fact  that  the  lessened  solu- 
bility is  the  same  whether  the  extraction  is  made  immediately 
after  mixing  the  floats  with  the  manure  or  after  allowing  the 
floats-manure  mixture  to  ferment  for  several  months.  It  might 
be  suspected  that  the  alkalinity  of  the  manure  could  have  been 
sufficient  to  neutralize  enough  of  the  acid  used  in  the  extraction 
to  account  for  the  weakened  action  of  the  floats,  in  the  floats- 
manure  mixture.  In  order  to  test  this  point,  portions  of  the  same 
extracts  as  used  for  the  analyses  reported  in  Table  IV,  were 
titrated  with  standard  alkali.  The  citric  acid  solution  used  to 
extract  the  mixture  of  floats  and  quartz  had  retained  83  per 
cent  of  its  original  strength;  that  used  to  extract  the  mixture 
of  floats,  manure  and  quartz  had  retained  75  per  cent  of  its 
original  strength.  In  these  cases  only  100  c.  c.  of  acid  had  been 
used  to  extract  100  g.  of  material.  The  weakening  of  the  acid 
due  to  the  alkalinity  of  the  manure  thus  seems  entirely  too 

small  to  account  for  a lowering  of  the  solvent  action  by  several 
times. 

That  the  particles  of  floats  adhere  to  and  are  held  by  the 
fragments  of  manure  seems  very  probable,  for  on  taking  sam- 
ples of  the  mixtures  and  shaking  them  with  water  in  a flask, 
and  then  letting  stand,  no  separation  of  floats  is  seen  to  take 
place.  Just  as  soon  as  the  organic  matter  is  partly  oxidized 
with  hydrogen  peroxide,  the  floats  settle-out  and  can  be  readily 
seen  on  the  bottom  of  the  flask.  It  seems  quite  possible  that  the 
moist  manure  covers  the  particles  of  floats  with  slimy  films, 


The  Availability  of  Rock  Phosphate  35 

which  the  dilute  citric  acid  penetrates  slowly.  It  also  seems 
probable  that  the  floats  made  soluble  by  the  fermenting  manure, 
may  be  precipitated  by  the  alkaline  medium,  resulting  in  the 
formation  of  fine  particles  in  contact  with  the  manure.  Of  all 
the  particles  of  floats  in  the  mixture,  these  fine  ones  precipitated 
in  intimate  contact  with  the  manure  would  probably  be  the 
most  efficiently  protected  from  a weak  solvent.  If  this  has 
been  the  case,  then  it  seems  that  the  oxidation  method,  as  per- 
formed, should  prove  successful.  The  difficulty,  however,  is 
that  the  extraction  dissolves  so  large  a quantity  of  phosphates 
from  the  floats  which  have  not  been  acted  upon  by  the  manure, 
as  to  entirely  mask  from  definite  chemical  measurement  any  sol- 
vent action  that  may  have  taken  place.  This  becomes  more 
apparent  when  we  consider  the  fact  that  in  the  analyses  of  the 
citric  acid  extractions  made  after  previous  oxidation,  as  re- 
ported in  Table  II,  the  amount  of  solution  used  fo,r  analyses 
represented  the  action  of  0.2  g.  of  manure  on  0.05  g.  floats.  Per- 
haps after  a much  longer  period  of  fermentation  it  will  be  pos- 
sible to  demonstrate  a definite  solvent  action  by  some  one  of  the 
methods  already  described.  While  the  results  thus  far  reported 
generally  favor  a slight  solvent  action,  the  amounts  are  far 
too  small  on  which  to  base  definite  conclusions. 

Solvents  Produced  by  Fermenting  Manure 

The  statement  is  frequently  made  that  rotting  manure  devel- 
ops various  organic  acids  that  help  to  bring  plant  food  mater- 
ial into  solution.  The  fact  that  the  fermenting  manure  and 
June  grass  used  in  the  foregoing  work  became  alkaline  after 
four  to  five  months  of  fermentation,  would  seem  to  indicate  that 
sufficient  bases  aye  present  during  the  early  stages  of  decom- 
position to  neutralize  any  free  acids  other  than  carbon  dioxide 
that  might  be  formed.  When  manure  is  applied  to  cultivated 
soils,  under  conditions  as  obtained  in  ordinary  farm  practice, 
it  disappears  quite  rapidly,  indicating  very  little  formation  of 
the  more  or  less  inert  humic  acids  and  humus  compounds.  How- 
ever, as  the  following  references  indicate,  the  addition  of  manure 
to  a soil  greatly  increases  the  carbon  dioxide  production  of  that 
soil. 

Boussingault  and  Lewy9  have  shown  that  the  air  of  rich  soils 


9 Johnson,  How  Crops  Feed,  p.  219. 


36 


Wisconsin  Research  Bulletin  No.  20 


containing  considerable  organic  matter,  may  contain  many  times 
as  much  carbon  dioxide  as  the  air  of  soils  poor  in  organic  mat- 
ter. 

Working  with  a soil  which  had  not  received  manure  for  some 
time,  Stoklasa  10  was  able  to  show  that  the  addition  of  manure 
in  the  proportion  of  10  g.  manure  to  1000  g.  soil,  which  is  an 
application  of  about  12  tons  per  acre,  calculating  two  and  one 
half  million  pounds  per  acre  eight  inches,  more  than  doubled 
the  carbon  dioxide  production  of  the  soil  during  the  following 
32  day  period.  With  a soil  that  has  been  manured  more  fre- 
quently, the  same  addition  of  manure  still  increased  the  carbon 
dioxide  production  appreciably. 

Sewerin11  has  shown  that  an  unsterilized  soil  may  produce 
ten  to  twenty  times  as  much  carbon  dioxide  as  a sterilized  soil. 

The  application  of  manure  to  a soil  serves  not  only  to  inocu- 
late that  soil  with  much  active  bacterial  life,  but  also  to  furnish 
food  and  improve  the  conditions  for  that  growth.  The  sum  total 
of  this  effect  greatly  increases  the  carbon  dioxide  production 
of  the  soil,  since  it  has  its  origin  in  the  respiration  of  the  soil 
life  and  the  oxidation  of  the  organic  matter. 

It  seems  reasonable  to  believe  that  the  solvent  action  of  ma- 
nure when  applied  to  normal  soils  is  largely  due  to  the  increased 
carbon  dioxide  production.  In  the  mixtures  of  organic  matter 
used  in  the  foregoing  work,  it  appears  that  carbon  dioxide  was 
the  only  free  acid  formed  and  hence  wrould  be  the  only  acidic 
substance  that  could  have  exerted  a solvent  action  on  the  floats. 
It  is  interesting  to  note  that  the  solvent  action  ascribed  to  bac- 
terial activity  is  often  much  greater  when  carbohydrates  are 
present  furnishing  proper  material  fo,r  the  production  of  car- 
bon dioxide.12  Since  the  addition  of  manure  favors  bacterial 
activity  it  is  probably  also  accompanied  by  increased  action  on 
insoluble  phosphates  due  to  the  enzymes  produced  by  the  bac- 
teria.13 Bacteria,  of  course,  use  up  soluble  phosphates  in  their 
own  metabolism,  and  thus  their  growth  may  be  followed  by  a 
decrease  in  soluble  phosphates  for  the  time  being,  as  Sewerin  ld 
has  shown.  We  must,  however,  not  fail  to  recognize  that  the 


io  Centbl.  Bakt.,  2 Abt.,  1911,  29,  p.  408. 
i;  Centbl.  Bakt.,  2 Abt.,  1910,  28,  p.  561. 

12  Mich.  Exp.  Sta.  Spec.  Bui.  43,  1908,  pp.  28-29. 
is  Stoklasa.  Centbl.  Bakt.,  2 Abt.  1911,  29,  p.  499. 
i4  Centbl.  Bakt.,  2 Abt.,  1910,  28,  p.  561. 


The  Availability  of  Rock  Phosphate 


37 


most  productive  soil  conditions  are  generally  accompanied  by 
intensive  bacterial  activity,  indicating  that  the  two  go  hand  in 
hand. 

Hopkins 15  suggests  that  the  nitric  acid  formed  in  the  pro- 
cess of  nitrification  may  act  on  insoluble  phosphates  and  make 
the  material  available  to  growing  crops.  Where  the  fermenting 
material  becomes  alkaline  as  was  the  case  in  the  present  work, 
the  nitric  acid  would  be  neutralized  as  soon  as  formed  and  hence 
could  not  act  on  the  floats.  A determination  of  nitrates  made 
on  the  fermenting  manure  mixtures  used  in  the  foregoing  in- 
vestigations gave  141  parts  of  nitrates  per  million  of  the  dry 
material,  showing  that  even  though  active  nitrification  was  tak- 
ing place,  the  nitric  acid  formed  was  insufficient  to  neutralize 
all  the  alkalinity  developed  at  the  same  time. 


Experiment  III  Effect  of  Manure  on  the  Carbon  Dioxide 
Production  of  Soils 

In  order  to  determine  how  the  manure  used  in  Experiment  II 
might  affect  the  carbon  dioxide  production  of  a soil,  the  follow- 
ing experiment  was  undertaken:  Four  2-gallon  glazed  earthen- 
ware jars  were  used.  Each  was  provided  with  a bent  glass  tube 
that  passed  through  the  center  of  the  bottom  of  the  jar.  The 
connections  of  the  tubes  with  the  jars  were  made  by  means  of 
rubber  stoppers  and  sealing  wax.  A small  perforated  tin  cover 
was  placed  over  the  opening  of  each  tube  inside  the  jars.  One 
kilo  of  gravel  consisting  of  particles  about  the  size  of  small  peas 
was  placed  in  each  jar,  just  covering  the  perforated  covers.  On 
top  of  this  gravel  7 kg  of  soil  were  placed.  To  the  soil  used  in 
two  of  the  jars,  200  g.  of  manure  were  previously  added  and 
mixed.  The  soil  used  was  sandy  and  low  in  organic  matter. 
The  manure  used  was  air-dried  cow  manure,  similar  to  that  used 
in  experiment  II. 

To  each  jar  800  c.  c.  of  water  were  added  and  the  jars  weighed. 
The  jars  were  placed  in  the  plant  house  and  contents  stipred 
and  watered  occasionally.  After  several  weeks,  active  fermen- 
tation was  taking  place  where  manure  had  been  added. 


is  Soil  Fertility  and  Permanent  Agriculture,  p.  197. 


38 


Wisconsin  Research  Bulletin  No.  20 


Figure  4.  Apparatus  for  determining  the  carbon  dioxide  production  of  soils 


Tiie  Availability  of  Rock  Phosphate 


39 


The  jars  were  watered  to  their  original  weight.  A glass  cover 
provided  with  a hole  was  fitted  into  the  top  of  each  jar.  The 
cover  was  held  in  place  about  three  inches  from  the  surface  of 
the  soil  by  means  of  a ring  support  passing  around  the  inside 
edge  of  the  jar  resting  on  supports  passing  to  the  bottom  of 
the  jar.  The  cover  was  sealed  to  the  jar  with  putty  and  the 
jars  covered  with  a coat  of  .varnish,  making  the  whole  air  tight. 
The  hole  in  the  cover  was  provided  with  a rubber  stopper  car- 
rying a bent  glass  tube.  This  tube  was  connected  to  a guard 
bottle  containing  sulphuric  acid,  and  this  to  another  containing 
caustic  potash.  A train  of  apparatus  as  ordinarily  used  in  the 
determination  of  carbon  dioxide  was  attached  to  the  tube  lead- 
ing from  the  bottom  of  the  jar.  Figure  4 shows  the  entire  ar- 
rangement of  apparatus  used. 

After  testing  for  leaks,  a stream  of  air  was  drawn  through 
until  the  apparatus  wTas  practically  freed  of  carbon  dioxide.  A 
20-liter  aspirator  was  used.  After  standing  12  hours  a slow 
stream  of  air  was  drawn  through  with  Geissler  bulb  in  place, 
till  the  carbon  dioxide  was  practically  all  drawn  out  of  the  jar. 
The  increase  in  weight  of  the  Geissler  bulb  thus  gave  the  weight 
of  carbon  dioxide  formed  in  the  jar  from  the  time  it  had  been 
freed  of  that  gas.  It  is  important  to  note  that  there  was 'suf- 
ficient air  in  the  jars  to  amply  supply  the  oxygen  needed  during 
a twelve-hour  period,  when  no  air  was  drawn  through.  In  the 
pore  space  of  the  soil  and  above  it  to  the  cover  there  were  four 
to  five  liters  of  air,  containing  sufficient  oxygen  for  the  produc- 
tion of  several  times  as  much  carbon  dioxide  as  formed.  The 
temperature  during  the  determination  was  that  of  the  labora- 
tory, about  20°  C.  The  results  of  this  work  are  given  in  Table 
V calculated  to  twenty-four-hour  periods. 


Table  V Production  of  Carbon  Dioxide  in  Manured  and  Unma- 
nured Soils 

Grams  of  Carbon  Dioxide  Formed  in  24  Hours 


Trial 

Unmanured  7 kg.  soil 

Manured  7 kg.  soil, 
200  g . manure 

Increase 
due  to 

.Tar  1 

Jar  2 

Av. 

Jar  3 Jar  4 

Av.  I 

manure 

1 

.1093 

.1590 

.1341 

.8559  .7567 

.8063 

.6722 

2 

.0864 

.1530 

.1197 

.7219  .7811 

.7515 

.6318 

40 


Wisconsin  Research  Bulletin  No.  20 


The  data  in  this  table  show  that  the  manured  soil  produced 
about  six  times  as  much  carbon  dioxide  as  the  unmanured  soil. 
The  200  g.  of  air  dried  manure  used,  represented  about  800  g. 
of  fresh  cow  manure.  This  800  g.  of  manure  to  7 kg.  of  soil 
represents  an  application  of  about  150  tons  pe,r  acre  if  we  as- 
sume  that  the  manure  is  mixed  with  the  surface  eight  inches, 
and  that  this  eight  inch  layer  over  an  acre  weighs  2,500,000 
pounds.  Calculating  from  these  data,  an  application  of  twenty- 
five  tons  of  manure  per  acre  would'  suffice  to  just  double  the  car- 
bon dioxide  production  of  the  surface  eight  inch  layer  of  a soil 
such  as  used.  One  kilo  of  the  unmanured  soil  produced  carbon 
dioxide  at  the  rate  of  about  18  mg.  in  24  hours,  and  calculating 
from  the  increase  due  to  the  manure,  10  g.  of  the  wet  manure 
produced  8 mg.  of  carbon  dioxide  in  24  hours. 

Stoklasa  16  working  with  a soil  low  in  organic  matter,  found 
that  1 kg.  produced  14  mg.  in  twenty-four  hours.  By  adding 
10  g.  of  fresh  cow  manure  to  1 kg.  of  soil,  the  carbon  dioxide 
production  was  just  about  doubled.  This  represents  an  appli- 
cation of  about  12  tons  per  acre,  calculating  on  the  same  basis 
as  before. 

Considering  that  the  two  experiments  were  carried  out  with 
different  manures  and  soils  in  different  proportions  and  by 
methods  considerably  different,  it  seems  remarkable  that  the 
results  should  concord  so  well.  Had  a smaller  proportion  of 
manure  to  soil  been  used  in  the  present  work,  as  Stoklasa  did, 
the  amount  of  carbon  dioxide  arising,  due  to  the  addition  of  10 
g.  of  manure  would  undoubtedly  have  been  larger  and  more 
nearly  equal  to  Stoklasa *s  figure. 


Experiment  IV.  Solvent  Action  on  Floats  of  Soil  Air 
from  Manured  and  Unmanured  Soils 

Tn  order  to  demonstrate  that  the  carbon  dioxide  production 
of  the  manured  soil  had  been  increased  sufficiently  through  the 
addition  of  the  manure  to  bring  about  a measurable  solvent 
action  on  floats,  the  following  experiment  was  performed  with 
the  set  of  jars  used  in  Experiment  III. 


10  Centbl.  Bakt.,  2 Abt.,  1911,  20,  p.  408. 


The  Availability  of  Rock  Phosphate 


41 


The  apparatus  with  jar  connected  having  been  freed  of  car- 
bon dioxide  twelve  hours  since,  a battery  of  four  Erlenmeyer 
flasks  was  attached  in  position  in  place  of  the  Geissler  bulb. 
Each  flask  contained  one-half  gram  of  floats  and  200  c.  c.  of 
water  freed  of  ammonia  and  carbon  dioxide.  Forty  liters  of  air 
we,re  then  drawn  through  by  means  of  an  aspirator,  the  ar- 
rangement being  such  that  the  passing  air  bubbled  through  the 
liquid  in  the  battery  of  flasks.  The  solutions  in  the  four  flasks 
were  poured  together,  filtered  and  analyzed  for  phosphates. 
The  battery  of  flasks,  containing  a fresh  supply  of  floats  and 
water  was  again  placed  in  position  as  before  and  two  liters  of 
air  drawn  through  twice  per  day  during  four  successive  days. 
The  solutions  were  filtered  and  analyzed  as  before.  Table  VI 
gives  the  results  of  this  work. 

Table  VI  Phosphates  Dissolved  by  Cabbon  Dioxide  from  Ma- 
nured and  Unmanured  Soil 


Mg'.  P2O5  dissolved  by 

Air  from  Soil 

(a) 

One  aspiration  of 
40  liters. 

‘ 

(b) 

8 successive  aspir- 
ations of  2 liters 
each. 

Tin  m n.nnrprl  

0.52 

1.08 



1.04 

2.20 

These  data  show  conclusively  that  the  addition  of  manure  to 
a soil  may  increase  the  carbon  dioxide  production  of  that  soil 
to  such  an  extent  that  an  increased  solvent  power  of  the  soil 
air  on  floats  is  readily  demonstrated.  The  addition  of  the  ma- 
nure has  just  about  doubled  the  solvent  power  under  the  condi- 
tions as  obtained. 

By  this  method  it  is  possible  to  show  that  fermenting  manure 
during  a period  of  a few  hours,  gives  rise  to  sufficient  carbon 
dioxide  to  measurably  exert  a solvent  action  on  floats.  With 
the  methods  used  in  the  previous  work,  little  or  no  action  could 
be  measured  after  four  or  five  months.  This  apparent  incon- 
sistency will  be  taken  up  in  the  final  discussion. 


42 


Wisconsin  Research  Bulletin  No.  20 


AVAILABILITY  OF  FLOATS  AS  INFLUENCED  BY 
THOROUGHNESS  OF  MIXING 

Experiment  V.  Influence  of  Thoroughness  of  Mixing 

For  the  purpose  of  determining  what  effect  thoroughness  of 
mixing  of  the  floats  with  the  soil  medium  might  have  on  the 
availability  of  the  phosphates  to  a growing  crop,  the  following 
experiment  was  carried  out.  A set  of  four-gallon  glazed  earth- 
enware jars  were  filled  with  50  pounds  of  quartz  and  treated  as 
follows : 


Set  No. 

. Treatment 

2 

D 1 n y»  1 -r- 

rSlcLIlK. 

15  grams  rock  phosphate  ordinarily  mixed 
k>  grams  rock  phosphate  thoroughly  mixed. 
7|  grams  rock  phosphate  ordinarily  mixed 
7t  grams  rock  phosphate  thoroughly  mixed. 

ol)  grams  acid  phosphate. 

15  grams  acid  phosphate. 

7|  grams  acid  phosphate. 



3i  grams  acid  phosphate. 

Tins  set  was  arranged  in  duplicate.  The  quartz,  floats,  and 
acid  phosphate  were  similar  to  the  materials  used  in  Experiment 
II.  In  order  to  secure  what  is  called  thorough  mixing  of  the 
floats  with  the  quartz,  the  floats  were  first  carefully  mixed  with 
1 kg.  of  quartz  and  then  this  mixture  with  the  remainder  of  the 
50  pounds  of  quartz.  In  the  case  of  what  is  called  ordinary 
mixing,  the  floats  were  sprinkled  on  the  surface  of  the  dry 
quartz  and  then  mixed  with  a suitable  instrument.  The  acid 
phosphate  was  similarly  applied. 

On  March  7 the  jars  were  planted  to  com.  Distilled  water 
was  used  throughout  the  experiment.  After  the  com  was  up 
and  growing  well,  it  was  thinned  to  three  plants  per  jar.  A 
nutrient  solution  containing  the  essential  elements  except  phos- 
phorus was  applied  once  a week.  It  was  deemed  more  prefer- 
able to  apply  this  frequently  in  small  quantities  diluted  with  a 
large  amount  of  water,  than  all  at  once  in  the  beginning,  since 
a large  quantity  of  salts  in  solution  may  of  itself  exert  a con- 
siderable solvent  action  on  floats.  The  jars  were  kept  in  the 
green  house  and  contents  watered  and  stirred  when  needed. 

TTp  to  April  1,  when  the  com  had  reached  a height  of  about 
15  inches,  the  corn  in  all  the  jars  except  the  blank  and  that 


The  Availability  of  Rock  Phosphate 


43 


receiving  30  g.  acid  phosphate  was  of  about  equal  appearance 
and  growing  well.  Where  30  g.  of  acid  phosphate  we, re  used, 
the  excess  of  acidity  seemed  to  be  injurious.  After  the  fourth 
week  the  corn  in  the  jars  where  the  floats  had  been  thoroughly 


2 4 6 8 10 

Blank  Ordinary  Thorough  Ordinary  Thorough 

Mixing  Mixing  Mixing  Mixing 

15  grams  Rock  Phosphate  7.5  grams  Rock  Phosphate 

Figure  5.  The  effect  on  the  growth  of  corn  of  thoroughness  of  mixing  rock  phosphate 
with  the  soil  medium. 


mixed  rock  phosphate. 

mixed,  was  beginning  to  show  a better  growth  than  that  where 
the  floats  were  ordinarily  mixed.  That  on  the  acid  phosphate, 
except  where  30  g.  were  used,  was  beginning  to  show  a slight 
advantage  over  all  the  rest.  Pictures  were  taken  April  29,  just 
as  the  corn  was  beginning  to  tassel  out.  (See  Figures  5 and  6). 


44 


Wisconsin  Research  Bulletin  No.  20 


On  May  22  the  c,rop  was  harvested.  Some  of  the  corn  had  form- 
ed small  nubbins. 

The  roots  were  removed  and  separated  from  the  quartz  with  a 
sieve,  returning  the  quartz  to  the  respective  jars. 

On  July  2 the  jars  were  planted  to  oats,  20  seeds  being  plant- 
ed in  each  jar.  The  oats  were  watered  and  given  nutrient  solu- 
tion as  in  the  case  of  the  corn.  The  oats  on  the  acid  phosphate 
made  a decidedly  better  growth  almost  from  the  beginning  than 
that  on  the  rock  phosphate.  That  on  the  thoroughly  mixed  rock 
phosphate  seemed  to  be  somewhat  better  on  the  average  than 
that  on  the  ordinarily  mixed.  On  August  3 the  oats  were  cut. 
They  had  headed  out  and  formed  light  kernels. 

Table  VII  gives  dry  weights  of  the  corn  tops  and  roots  and 
oat  tops  as  secured  in  the  foregoing  work. 


Table  VII  Effect  of  Different  Phosfhate  Treatments  on  the 
Yield  of  Corn  and  Oats 


Set 

No. 

Treatment 

Corn 

Oats 

Tops 

Roots 

Av.  dry 
wt.  total 
crop. 

Tops 

a 

b 

! av. 

a 

b 

av. 

a 

b 

av. 

2 

Blank 

i 

0 8 

: 

2-0 

4 

15  g.  floats  ord.  mix 

51.8 

35.8 

43.8 

10.1 

6.6 

8.3 

52*1 

7.0 

7.2 

7.1 

G 

15  g-.  floats  thoro.  mix. . 

57.8 

59.0 

58.4 

14.7 

10.6 

12.6 

71.0 

11.8 

6.5 

9.1 

8 

7i  g.  floats  ord.  mix 

44.5 

37.4 

40.9 

8.4 

8.8 

8.6 

49.5 

4.3 

4.7 

4.5 

10 

7i  g.  floats  thoro.  mix... 

57.8 

56.7 

57.2 

13.1 

10.7 

11.9 

69.1 

6.4 

7.2 

6.8 

12 

30  g.  acid  phosphate 

24.8 

19.4 

22.1 

5.7 

3 4 

4.5 

26.6 

12.5 

13.8 

13.1 

14 

15  g.  acid  phosphate 

73.0 

58.5 

65.7 

11.5 

13.1 

12.3 

78.0 

24.0 

19.0 

21.5 

16 

7i  g.  acid  phosphate 

71.9 

63.0 

67.4 

12.4 

9.9 

11.1 

78.5 

17.0 

18.4 

17.7 

18 

3§  g-.  acid  phosphate 

68.0 

66.5 

67.2 

10.4 

14.1 

12.2 

79.4 

16.2 

16.5 

16.3 

From  this  table  and  Figure  5 it  is  evident  that  thoroughness 
of  mixing  had  a decided  effect  on  the  crop.  In  the  case  of  the 
corn  the  yield  with  thorough  mixing  approaches  quite  close  to 
the  yield  with  acid  phosphate.  When  30  g.  of  acid  phosphate 
were  used,  the)  excess  of  acidity  greatly  reduced  the  yield. 
Although  the  quartz  was  thoroughly  sifted  and  mixed  in  order 
to  remove  the  corn  roots,  jyet  on  the  average  the  effect  of  the 
thorough  initial  mixing  is  still  apparent  on  the  oat  crop.  The 
yield  of  oats  with  floats  is  far  behind  the  yield  with  acid  phos- 
phate, indicating  that  oats  have  a weaker  feeding  power  for  rock 
phosphates  than  corn.  However,  the  cases  are  not  strictly  com- 
parable, since  the  corn  growing  first  had  a chance  to  use  up  the 
more  easily  soluble  phosphates  in  the  floats. 


The  Availability  of  Rock  Phosphate  45 

Since  the  jars  contained  no  organic  matte, r or  carbohydrates 
of  any  kind,  it  does  not  seem  probable  that  the  floats  were  made 
soluble  through  bacterial  activity.  Had  the  floats  been  made 
soluble  by  any  other  means  than  the  plants  themselves,  then 
this  soluble  portion  would  have  become  distributed  throughout 
the  contents  of  the  jars,  and  then  the, re  should  not  be  this  large 
difference  between  ordinary  and  thorough  mixing.  It  seems 
more  reasonable  to  believe  that  the  plants  themselves  exert  a 
marked  solvent  action  on  floats.  Since  all  attempts  to  find  ap- 
preciable amounts  of  acid  root  excretions  other  than  carbon 
dioxide  have  failed,  this  solvent  action  must  be  attributed  to  the 
carbon  dioxide  given  off  by  the  roots. 

Importance  of  the  Carbon  Dioxide  Production 
of  Plant  Roots 

Stoklasa 17  has  measured  the  carbon  dioxide  production  of 
roots  of  various  plants.  Calculated  to  dry  weight,  he  found  that 
100  g.  of  twenty-five-day-old  wheat  roots  produced  2.54  g.  of  car- 
bon dioxide  in  24  hours.  Similarly,  fifty-day-old  clover  roots 
produced  5.8  g.  of  carbon  dioxide.  From  his  data  obtained  dur- 
ing the  whole  vegetative  period,  he  calculated  tha"  the  average 
carbon  dioxide  production  of  wheat  roots  per  day  for  the  vege- 
tative period  is  60  kg.  per  ha. 

Kossowitsch18  calculated  from  his  data  that  the  roots  of  one 
ha.  of  mustard  plants  produce  during  the  vegetative  period 
2250  kg.  of  carbon  dioxide.  Assuming  the  following  reaction: 
Ca3  (P04)2  plus  4 C02  plus  4 H20  equals  2 Ca  (HC03)2  plus 
CaH4(P04)2,  he  Calculates  from  the  analyses  of  the  mustard 
plants  that  twenty  times  as  much  carbon  dioxide  is  produced  as 
is  required  to  bring  the  phosphates  used  by  the  plants  into  solu- 
tion. 

Theoretically,  according  to  the 'reaction  above,  1.24  parts  o(‘ 
carbon  dioxide  dissolve  one  part  of  phosphoric  anhydride. 

Krober,19  calculating  from  the  phosphates  brought  into  solu- 
tion by  the  carbon  dioxide  formed  by  cultures  of  yeast,  found 
that  it  required  sixty-eight  to  sixfy-nine  parts  of  carbon  dioxide 
to  bring  one  part  of  phosphoric  anhydride  into  solution.  From 

17  Centbl.  Bakt.  2 Abt.,  1905,  14,  p.  735. 

is  Russ.  Jour.  Exp.  Landw.  1904,  4,  p.  493. 

is  Jour.  Landw.,  1909,  57,  p.  38. 


46  Wisconsin  Research  Bulletin  No.  20 

this  he  concluded  that  it  takes  much  larger  quantities  than  Kos- 
sowitsch  considered  necessary. 

Perhaps  the  reason  Krober  finds  that  such  large  amounts  of 
carbon  dioxide  are  necessary,  is  due  to  the  conditions  that  pre- 
vailed in  his  experiment.  The  volume  of  his  solution,  it  ap- 
pears, amounted  to  250  c.  c.  and  22.75  g.  of  carbon  dioxide  were 
developed  during  the  fermentation.  A liter  of  wate,r  will  dis- 
solve only  about  two  grams  of  carbon  dioxide  under  ordinary 
conditions.  As  long  as  the  water  is  saturated  any  additional 
carbon  dioxide  passes  off  and  does  not  affect  the  amount  of  a 
substance  that  will  be  brought  into  solution.  Had  his  volume 
of  solution  been  larger,  the  efficiency  of  the  carbon  dioxide  un- 
doubtedly would  have  been  increased. 

Calculating  from  the  average  results  of  various  investigators20 
it  is  found  that  in  carbon  dioxide  saturated  water,  at  ordinary 
conditions  of  temperature  and  pressure,  it  takes  about  six  parts 
of  carbon  dioxide  to  dissolve  one  part  of  phosphoric  anhydride 
from  precipitated  tri-calcium  phosphate.  To  dissolve  the  same 
amount  of  phosphoric  anhydride  from  rock  phosphate  takes  three 
to>  four  times  as  much  carbon  dioxide.  The  efficiency  of  the 
carbon  dioxide  in  bringing  phosphates  into  solution  depends  en- 
tirely on  the  conditions  under  which  the  carbon  dioxide  acts  and 
what  the  nature  of  the  insoluble  phosphates  are. 

It  is  most  important  to  recognize  that  the  carbon  dioxide  given 
off  by  the  plant  roots  exercises  its  solvent  action  under  condi- 
tions which  have  never  been  imitated  in  the  laboratory.  The  re- 
action proceeding  when  carbon  dioxide  acts  on  phosphates  must 
be  considered  as  of  the  nature  of  a balanced  action.  The  plant 
immediately  absorbing  the  portion  made  soluble,  the  reverse  re- 
action is  prevented  and  hence  the  carbon  dioxide  works  under 
a maximum  efficiency  in  bringing  phosphates  into  solution.  How 
efficient  a solvent  carbon  dioxide  is  for  phosphates  under  these 
plant  root  conditions  is  still  a matter  of  mere  conjecture.  Per- 
haps the  best  guess  that  can  be  made  is  that  the  efficiency  lies 
somewhere  between  the  theoretical  and  that  which  is  obtained 
by  the  methods  now  used  in  the  laboratory. 

From  the  foregoing  considerations,  it  seems  probable  that  be- 
cause of  the  carbon  dioxide  given  off  by  the  roots,  plants  are  able 
to  secure  a large  portion  of  their  phosphate  supply  from  inso- 


20  COmey,  Diet.  Chem.  Sol’s,  p.  298. 


The  Availability  of  Rock  Phosphate 


47 


luble  sources,  provided  the  insoluble  sources  are  well  distributed 
and  thus  bring  particles  in  near  contact  to  the  whole  root  system 
of  the  growing  plant.  It  is  now  becoming  quite  generally  be- 
lieved that  the  efficiency  of  acid  phosphate  is  due  not  to  the  fact 
that  the  phosphate  remains  more  soluble,  but  to  the  fact  that 
the  phosphate  becomes  distributed  quite  thoroughly  through 
the  feeding  area  of  the  soil.  This  seems  all  the  more  probable 
from  the  general  recognition  that  soluble  phosphates  are  usually 
soon  precipitated  from  solution  when  applied  to  a soil. 


General  Discussion 

It  seems  reasonable  to  believe  that  in  most  cases  any  factor 
which  will  aid  in  giving  better  distribution  of  the  rock  phosphate 
through  the  soil  or  aid  in  bringing  the  phosphate  in  more  general 
contact  with  the  plant  roots,  will  result  in  making  that  phos- 
phate more  available  to  the  growing  crop.  If  this  conception  of 
availability  is  accepted,  then  it  is  apparent  that  for  the  subject 
under  consideration,  availability  as  measured  by  a weak  solvent 
may  be  entirely  different  from  availability  as  measured  by  a 
growing  crop.  Moreover,  it  is  to  be  recalled  that  the  mere  mix- 
ing of  floats  with  manure  resulted  in  the  floats  becoming  less 
available  when  availability  is  measured  by  the  0.2  per  cent  citric 
acid  solution.  Yet,  the  numerous  field  experiments  point  quite 
conclusively  to  the  belief  ^that  this  practice  makes  the  rock  phos- 
phate more  available  to  growing  crops.  * 

Since  it  is  easyd^tIembnstratev'ThafTlie  addition  of  manure  to  a 
soil  results  in  a large  increase  of  the  carbon  dioxide  production  of 
that  soil,  and  this  increase  in  carbon  dioxide  production  can  be 
shown  to  result  in  an  increased  solvent  action  on  floats,  it  seems 
reasonable  to  conclude  that  fermenting  manure  does  exert  a sol- 
vent action  on  floats  which  are  in  contact  with  the  manure.  Since 
the  laboratory  composting  experiments  fail  to  measure  this  sol- 
vent action  satisfactorily,  it  only  remains  to  be  said  that  the 
methods  so  far  devised  for  these  experiments  are  not  suited  for 
the  purpose  at  hand. 

We  must  not  fail  to  recognize  that  the  conditions  in  the  labor- 
atory composting  experiments  are  far  different  from  the  condi- 
tions obtained  in  the  field,  where  mixed  manure  and  floats  are 
applied.  In  the  field  the  movements  of  soil  water  and  the  pres- 
ence of  nearby  growing  plant  roots  are  constantly  at  work  re^ 


48 


Wisconsin  Research  Bulletin  No.  20 


moving  from  the  particles  of  manure  in  contact  with  floats,  the 
phosphates  made  soluble.  Under  such  conditions,  as  already  ex- 
plained a solvent  action  may  progress  under  maximum  effic- 
iency. the  phosphates  made  soluble,  if  not  used  immediately 
by  a growing  crop,  are  distributed  by  the  movements  of  the  soil 
water  and  precipitated  in  fine  pa,rticles  over  a large  area  in  fit 
condition  to  be  used  by  succeeding  crops. 

With  the  composts  used  in  the  laboratory  experiments  the 
soluble  portion  is  not  removed  from  the  sphere  of  action  and 
hence  the  solvent  power  may  be  checked  as  soon  as  the  particular 
solvent  becomes  saturated.  The  efficiency  of  the  carbon  dioxide 
formed  by  the  fermenting  manure  in  bringing  phosphates  into 
solution  depends  upon  the  rate  at  which  the  already  soluble  por- 
tion is  removed,  and  upon  the  amount  of  water  present.  When 
the  water  present  becomes  saturated  with  carbon  dioxide,  and 
the  phosphates  already  in  solution  are  not  removed,  any  further 
production  of  carbon  dioxide  is  dissipated  to  the  atmosphere  and 
does  not  aid  in  bringing  phosphates  into  solution.  Accepting 
this  conception  of  the  conditions  that  prevail  in  the  laboratory 
composting  experiments,  then  these  experiments  should  reveal 
a slight  solvent  action,  and  this  is  just  what  they  do  on  a gen- 
era!  average. 

It  seems  that  the  mixing  of  floats  with  manure  should  result 
m favorable  conditions  for  the  influence  of  bacterial  activity  on 
the  floats.  Each  particle  of  manure  in  the  soil  undoubtedly  be- 
comes a center  of  increased  active  bacterial  life,  'and  if  floats 
are  in  contact  with  the  manure,  the  chances  for  bacterial  action 
on  the  floats  are  greatly  increased.  Although  the  bacteria  may 
use  up  the  phosphates  in  the  floats  in  their  own  metabolism,  yet 
ultimately  after  death  and  decay  of  these  bacterial  cells,  the 
phosphates  contained  therein  will  have  become  more  finely  divid- 
ed and  better  distributed,  resulting  in  an  increased  availability 
to  succeeding  crops. 

Undoubtedly  the  practice  of  thorough  mixing  of  the  floats 
with  manure  results  in  a better  initial  mechanical  distribution 
of  the  floats  with  the  soil  than  is  generally  obtained,  when  the 
floats  are  applied  directly  to  a soil.  To  thoroughly  mix  a pound 
of  floats  with  several  tons  of  soil  would  be  a difficult  matter  if 
the  floats  were  added  directly  to  all  of  the  soil.  If  the  floats 
w ore  mixed  with  a hundred  pounds  of  the  soil  and  then  this 
with  the  remaining  soil,  much  more  efficient  admixture  would  be 


The  Availability  of  Rock  Phosphate 


49 


possible.  To  a certain  extent,  this  same  factor  influencing  thor- 
oughness of  mixing  is  secured  when  floats  are  first  mixed  with 
manure,  previous  to  application.  The  direct  application  of  floats 
to  a tight  clay  soil  low  in  organic  matter  may  result  in  the  floats 
becoming  locked  up  in  local  areas  which  represent  a compar- 
atively small  portion  of  the  cultivated  layer.  Further  distri- 
bution either  physically  or  chemically  would  be  a slow  process 
in  such  instances.  The  use  of  manure  and  other  organic  mater- 
ials prevents  such  cases  as  this. 

It  is  a matter  of  common  occurrence  to  find  fragments  of  man- 
ure or  other  organic  materials  present  in  the  soil  completely  en- 
tangled with  plant  roots.21  The  roots  are  attracted  cliemotact- 
ically  by  the  soluble  plant  food  in  the  manure.  When  floats  are 
mixed  with  manure,  this  same  action  may  attract  the  roots  of 
the  growing  crop  to  the  centers  of  rock  phosphate  supply  and 
thus  to  a certain  extent  the  phosphate  is  made  more  available  to 
the  crop  because  of  the  manure. 

The  use  of  manure  and  crop  residues  favors  the  activity  of 
many  of  the  very  same  agencies  that  unlock  the  insoluble  phos- 
phates of  the  granites  and  make  them  available  to  growing  crops. 
Can  there  then  be  any  question  but  that  these  same  organic  sub- 
stances help  to  make  floats  available  to  growing  crops ! 

i 

Summary 

The  laboratory  experiments  in  which  organic  matter  is  com- 
posted with  raw  phosphates  reveal  on  a general  average  only  a 
slight  solvent  action  of  the  fermenting  material  on  the  phos- 
phates. The  amount  of  solvent  action  is  always  within  the  limit 
of  experimental  error.  Considering  the  conditions  that  prevail 
in  these  composting  experiments,  this  result  is  exactly  in  accord 
with  what  is  to  be  expectedf.for,  since  carbon  dioxide  is  the  only 
free  acid  formed  in  these  composts,  only  a slight  solvent  action 
should  be  measured.  The  amount  of  the  solvent  action  measured 
is  limited  to  the  amount  of  phosphate  which  the  carbon  dioxide- 
charged  water  can  hold  in  solution. 

In  the  composting  experiments,  the  dissolved  phosphates  and 
carbonates  are  not  removed  from  the  field  of  action,  and  hence 
the  reaction  bringing  phosphates  into  solution  quickly  , reaches  a 
state  of  equilibrium,  after  which  any  further  production  of  car- 


21  Hall — Fertilizers  and  Manures,  p.  290. 


50 


Wisconsin  Research  Bulletin  No.  20 


bon  dioxide  is  dissipated  to  the  atmosphere  and  aids  nothing  in 
bringing  phosphates  into  solution. 

Under  field  conditions  the  movements  of  soil  wate,r  and  the 
feeding  of  crops  are  constantly  removing  dissolved  phosphates 
and  carbonates  from  the  little  centers  of  solution,  existing  as 
fragments  of  organic  material  where  intensive  carbon  dioxide 
production  takes  place.  This  continual  removal  of  the  dissolved 
substances  results  in  conditions  under  which  the  efficiency  of  car- 
bon dioxide  as  a solvent  is  greatly  increased. 

Since  in  the  composting  experiments  the  dissolved  substances 
are  not  removed  as  under  -field  conditions,  it  must  be  concluded 
that  the  laboratory  experiments  fail  to  imitate  field  conditions 
with  regard  to  a most  vital  consideration. 

The  mere  mixing  of  floats  with  manure  makes  the  floats  less 
soluble  in  0.2  pe,r  cent  citric  acid  solution.  This  lessening  in 
solubility  takes  place  immediately  on  mixing  the  floats  with  the 
manure.  Prom  this  it  must  be  concluded  that  the  availability  of 
phosphates  as  measured  by  a solvent  like  0.2  per  cent  citric  acid 
may  be  entirely  different  from  availability  as  measured  by  a 
growing  crop. 

The  availability  of  raw  phosphates  as  measured  by  a growing 
crop  is  influenced  not  only  by  its  solubility  in  weak  solvents,  but 
also  to  a large  extent  by  the  thoroughness  with  which  it  is  dis- 
tributed through  the  feeding  area  of  the  soil. 

When  floats  are  thoroughly  mixed  through  the  feeding  area  of 
the  soil,  it  appears  that  some  species  of  plants  are  able  to  secure 
nearly  an  adequate  supply  of  phosphates-  from  the  insoluble 
floats.  It  seems  that  the  carbon  dioxide  given  off  by  the  plant 
roots  is  instrumental  in  bringing  the  phosphates  into  solution 
and  thus  making  the  floats  available. 

The  addition  of  manure  to  a soil  greatly  increases  the  carbon 
dioxide  production  of  that  soil.  It  is  easy  to  demonstrate  that 
this  increased  carbon  dioxide  production  exerts  during  a short 
period  a measurably  increased  solvent  action  on  floats.  'When 
floats  are  mixed  with  manure,  the  raw  phosphate  is  placed  at 
the  centers  of  carbon  dioxide  production  and  bacterial  activity, 
giving  ideal  conditions  for  solution  and  distribution  of  the  phos- 
phates. 

It  seems  that  the  addition  of  floats  to  a tight  clay  soil  low  in 
prganic  matter  may  result  in  the  floats  becoming  locked  up  in 


The  Availability  of  Rock  Phosphate 


51 


local  areas  from,  which  further  mechanical  and  chemical  distri- 
bution of  the  phosphates  would  be  very  slow. 

After  carefully  considering  the  more  important  factors  affect- 
ing the  availability  of  floats  to  growing  crops,  there  seems  to  be 
little  question  but  that  the  use  of  organic  matter  in  connection 
with  the  floats  increases  this  availability.  The  organic  matter 
brings  about  this  increased  availability  by  favoring  a more  effi- 
cient initial  mechanical  distribution  of  the  floats  with  the  soil 
and  by  favoring  the  .chemical  and  biological  processes  that  give 
rise  to  carbon  dioxide  and  other  agencies  which  attack  floats  and 
ultimately  give  the  material  a finer  and  more  uniform  distribu- 
tion through  the  soil. 


*21 

Studies  oi  the  Nutrition  of  the  Pig 


NOTES  ON  THE  CREATININ  EXCRETION  OF  THE  PIG 

E.  V.  McCOLLUM. 

With  the  discovery  by  Folin  that  the  amount  of  creatinin 
excreted  by  an  individual  is  nearly  constant,  together  with 
his  contribution  of  a quantitative  method  for  its  determin- 
ation, which  in  accuracy  and  ease  of  operation  leave  little  to 
be  desired,  we  are  in  possession  of  a valuable  aid  to  the  study 
of  certain  phases  of  protein  metabolism.  The  work  of  Folin16 
leaves  little  doubt  that  there  are  normally  two  kinds  of  pro- 
tein metabolism  going  on  simultaneously  in  the  animal  body. 
One  is  the  essential  or  endogenous  and  is  practically  constant 
and  represents  the  processes  of  cellular  activity.  The  other 
is  the  exogenous  which  is  exceedingly  variable  and  represents 
the  prompt  conversion  of  the  nitrogen  of  the  food  protein 
into  the  end  products  of  metabolism,  principally  urea.  The 
magnitude  of  this  type  depends  upon  the  protein  intake  and 
at  moderately  high  levels  is  proportional  to  it.  The  endo- 
genous type  results  in  the  formation  of  a distinct  group  of 
end  products,  the  only  one  of  which  we  have  any  definite 
knowledge  being  creatinin. 

Folin,  working  with  men,  did  not  attempt  to  eliminate  en- 
tirely the  exogenous  type  of  metabolism  by  long  continued 
feeding  of  a ration  very  low  in  nitrogen.  After  ten  days  on 
diets  containing  less  than  a gram  of  nitrogen  a day  the  men 
in  his  experiments  still  eliminated  more  than  60  per  cent  of 
the  total  nitrogen  of  the  urine  as  urea.  Since  urea  is  the 
nitrogenous  constituent  suffering  the  most  marked  diminu- 
tion in  quantity  as  the  exogenous  metabolism  is  diminished, 
it  did  not  appear  from  his  experiments  how  nearly  the  latter 


i6  Folin,  Amer.  Jour.  Physiol.,  13,  84  (1905). 


54 


Wisconsin  Research  Bulletin  No.  21 


type  had  been  eliminated.  In  order  to  arrive  at  a condition 
in  which  the  whole  of  the  protein  metabolism  is  of  the  endog- 
enous type,  provided  this  is  possible,  it  would  be  necessary 
to  keep  an  animal  during  a long  period  on  a diet  free  from 
nitrogen,  but  supplying  all  the  other  necessary  elements  and 
organic  complexes  necessary  to  normal  metabolism.  Doubt- 
less, also,  these  things  should  be  supplied  in  generous  but  not 
excessive  amounts,  and  the  animal  should  be  kept  under  con- 
ditions as  nearly  normal  as  possible.  These  conditions  are 
difficult  to  realize  with  any  animals  ordinarily  employed  in 
metabolism  studies.  A considerable  experience  with  the  pig 
as  a subject  in  our  work  with  the  mineral  elements  led  me 
to  the  belief  that  this  animal  would  be  unusually  valuable 
for  studies  in  protein  metabolism.  This  belief  was  strength- 
ened by  the  observation  that,  when  kept  in  a cage,  a vigorous 
pig  will  take  a sufficient  quantity  of  a solution  of  starch 
containing  the  necessary  salts,  to  meet  all  its  energy  require- 
ments, day  after  day,  with  no  evidence  of  anorexia  and  with 
no  appreciable  loss  in  weight.  In  an  experience  with  more 
than  a dozen  animals,  extending  over  a period  of  two  and  a 
half  years  I have  found  only  two  animals  which  proved  un- 
satisfactory in  this  respect. 

Preliminary  to  a series  of  experiments  in  which  it  was  de- 
sired to  feed  quantities  of  nitrogen  equivalent  to  the  endogen- 
ous nitrogen  metabolism  of  the  animal,  an  examination  was 
made  of  the  possibility  of  using  the  creatinin  excretion  as  an 
index  to  the  amount  of  nitrogen  derived  daily  from  this 
source.  It  was  believed  that  if  the  animals  were  given  a 
liberal  energy  supply  in  the  form  of  starch,  and  a salt  mix- 
ture containing  all  the  essential  radicals,  and  water,  he  would 
ultimately  reduce  his  exogenous  protein  metabolism  to  nearly, 
if  not  quite  the  vanishing  point.  In  this  condition  the  ratio 
of  creatinin  nitrogen  to  total  nitrogen  in  the  urine  should  be- 
come constant.  If  this  constant  ratio  should  be  confirmed  for 
a sufficient  number  of  animals  it  would  be  a very  valuable 
one.  Such  a ratio  would  make  it  possible  to  determine  the 
nitrogen  from  endogenous  metabolism.  It  would  only  be  nec- 
essary to  determine  the  creatinin  in  the  urine  for  a number  of 
days,  to  arrive  at  an  average  value,  and  multiply  the  nitrogen 


Notes  on  the  Creatinin  Excretion  of  the  Pig 


55 


appearing  in  this  form  by  the  factor  derived  from  the  con- 
stant ratio. 

The  results  of  experiments  with  seven  animals  are  re- 
corded in  Table  I.  Since  only  the  last  portions  of  the  rec- 
ords are  of  interest,  only  the  following  data  are  given.  Body 
weight;  length  of  the  period  on  a nitrogen-free  diet;  aver- 
age nitrogen  content  of  the  urine  during  the  last  five  days; 
average  content  of  creatinin  nitrogen,  and  its  ratio  to  the 
total  nitrogen.  It  will  be  seen  that  the  ratio  of  creatinin  nitro- 
gen to  total  nitrogen  finally  established  is  between  17.5  and 
19  to  100.  The  average  of  all  the  experiments  except  the 
last  is  18.5.  The  pig  showing  a ratio  of  22  to  100  had  been 
killed  before  the  results  were  calculated,  only  the  colorimeter 
readings  having  been  recorded  at  the  time  of  the  experiments 
and  the  calculations  made  at  a later  date.  It  was  therefore 
not  possible  to  try  this  pig  again.  However,  the  ratios  from 
the  other  animals  fall  within  so  narrow  a range  that  their 
average  18.5  may  be  safely  accepted  as  a close  approxima- 
tion to  the  final  ratio  on  the  level  where  the  metabolism  re- 
mains constant. 


TABLE  I.  creatinin  n.  and  total  n.  eliminated,  with  n.-free  diet 

The  pigs  were  kept  for  long  periods  on  N.-free  diet  and  the  ratio  of  Creatinin  N.  to 
Total  N.  eliminated  in  the  urine  was  found. 


No.  of  pig 

Initial 

weight 

lbs. 

Days 

on  N.-free 
diet. 

Av.  N.  con- 
tent of  urine 
last  5 days 

Av.  creatinin 
N.  in  urine 
last  5 days 

Per  cent  of 
total  N.  as 
creatinin 

16 

24 

27 

.54 

.104 

19.19 

21 

85 

24 

1.83 

.336 

18.36 

9 

150.5 

24 

2.65 

All 

18  05 

8 

43.5 

36 

1.09 

.193 

18.50 

5 

37 

21 

.90 

.160 

17.56 

10 

82 

24 

1.61 

.314 

19.17 

7 

165 

23 

2.61 

.574 

22.00 

The  animals  lost  but  little  weight  in  any  case,  and  in  a 
number  the  weight  remained  constant  throughout  the  ex- 
periment. The  time  required  to  attain  this  ratio  depends 
on  the  previous  state  of  nutrition  and  on  the  volume  of  urine 
eliminated.  A high  output  of  urine  tends  to  a more  rapid 
attainment  of  the  minimum  level  of  nitrogen  excretion.  I 
have  not  observed  the  appearance  of  the  above  constant  ratio 
in  any  case  before  the  sixteenth  day  on  a nitrogen-free  diet. 


Wisconsin  Research  Bulletin  No.  21 


56 


The  fact  that  a nearly  constant  ratio  of  creatinin  nitrogen  to 
total  nitrogen  is  established  under  the  conditions  of  these  ex- 
periments does  not,  of  course,  necessarily  mean  that  all  of  the 
nitrogen  in  the  urine  is  derived  from  endogenous  sources.  An 
examination  of  several  of  these  urinfes  showed  that  about  60 
per  cent  of  the  total  nitrogen  was  still  present  in  the  form 
ol!  urea. 

Urea  may  also  be  a product  of  endogenous  metabolic  pro- 
cesses, or  may  result  from  the  further  decomposition  of  un- 
known bodies  so  derived.  However  this  may  be,  the  nitrogen 
eliminated  when  this  nearly  constant  ratio  is  reached,  repre- 
sents the  absolute  minimum  level  of  protein  metabolism  of 
•which  the  animal  is  capable,  and  if  nitrogen  equilibrium  is  to 
be  maintained,  at  least  this  amount  of  nitrogen  must  be  sup- 
plied in  the  food  and  in  a utilizable  form.  We  are  justified 
in  assuming  this  nitrogen  to  be  derived  wholly  from  endo- 
genous sources  until  further  information  is  gained. 

If  we  find  the  average  amount  of  creatinin  nitrogen  ap- 
pearing in  the  urine  during  four  or  five  days  when  the  pig 
is  taking  no  food  from  animal  sources,  (that  is  a creatin-and 
creatinin-free  diet)  and  multiply  this  by  5.5,  the  product  will 
closely  approximate  the  amount  of  nitrogen  which  the  animal 
would  eliminate  daily  in  the  urine  if  kept  for  a long  period 
on  a nitrogen-free  diet.  It  is  safe  to  accept  this  as  the  main- 
tenance level. 

Whether  this  factor  will  also  apply  to  all  species  of  animals 
as  well  as  it  does  in  the  case  of  the  pig,  cannot  be  told  at  the 
present  time,  since  no  other  species  has  as  yet  been  investi- 
gated. Some  investigators  have  not  found  the  creatinin  excre- 
tion as  constant  in  the  carnivora  (dog)  as  has  been  found  by 
Folin  with  men  and  by  myself  with  the  pig.  Folin17  has 
suggested  that  the  endogenous  metabolism  of  the  carnivora 
may  not  be  as  constant  as  in  other  types  of  animals. 

Another  way  in  which  the  creatinin  excretion  will  with- 
out doubt  be  of  very  great  value  in  nutrition  studies  is  in 
serving  as  a basis  for  the  calculation  of  rations  in  animals 
employed  in  exact  nutrition  studies.  In  the  past  it  has  been 


n Folin,  Amer.  Jour,  physiol.  13,  84  (1905), 


Notes  on  the  Creatinin  Excretion  of  the  Pig 


57 


the  custom  of  investigators  to  base  the  calculation  of  rations 
on  the  body  weight  of  the  animal.  This  method  has  always 
been  known  to  have  little  merit,  because  of  the  great  varia- 
tion in  the  fat  content  of  the  body.  In  creatinin  we  have  an 
end  product  of  cellular  activity  and  which  is  eliminated  with 
surprising  regularity,  and  while  the  amount  eliminated  is 
only  roughly  proportional  to  the  body  weight,  it  seems  to  be 
as  Folin  pointed  out,  closely  proportional  to  the  amount  of 
metabolizing  tissue  in  the  body.  If  therefore,  it  is  desired  to 
feed  two  animals  comparable  quantities  of  a substance,  as 
protein,  it  would  undoubtedly  be  much  more  accurate  to  feed 
them  each  the  same  multiple  of  their  respective  creatinin 
nitrogen  excretion.  This  method  is  being  used  in  this  labor- 
atory in  work  now  in  progress  on  the  efficiency  with  which 
the  growing  pig  utilizes  certain  forms  of  nitrogen  for  growth. 

It  seemed  to  me  worth  while  to  study  the  relationship  be- 
tween the  rise  of  creatinin  elimination  and  the  retention  of 
nitrogen  during  growth  in  the  pig.  Data  on  this  point  have 
been  collected  with  only  three  pigs  and  is  presented  here 
(Table  II)  without  any  expression  as  to  finality  since  it  may 
need  revision  in  the  light  of  further  study. 


TABLE  II.  N.  RETENTION  AND  RISE  OF  CREATININ  ELIMINATION 
Relation  between  nitrogen  retention  and  the  rise  of  creatinin  elimination  in  growing 


Grams  N.  retained 

Grams  rise  in 
creatinin  N. 

Grams  N.  re- 
tained corre- 
sponding to  a rise 
of  1 mgm.  of 
creatinin  N, 

175 

.073 

.039 

.134 

2.39 
2.18  a 

2.40 

329 

a The  pig  was  sick  at  the  close  of  this  experiment  and  failed  one  day  to  void  anv 
urine,  dunng  the  last  six  days  from  which  the  final  creatinin  output  was  estimated 
This  disturbance  in  the  record  renders  the  figure  of  very  uncertain  value. 


58 


Wisconsin  Research  Bulletin  No.  21 


NATURE  OF  THE  REPAIR  PROCESSES 
OF  PROTEIN  METABOLISM 

f E.  V.  McCOLLUM. 

As  a result  of  the  great  advances  in  our  knowledge  of  the 
chemistry  of  the  proteins,  numerous  problems  relating  to  pro- 
tein metabolism  in  the  animal  have  arisen.  It  is  now  known 
that  proteins  from  various  sources  differ  widely  from  one  an- 
other in  fundamental  characters,  and  that  all  are  not  equiva- 
lent as  nutrients  for  animals.  Casein  and  vitellin  have  been 
shown  to  be  capable  of  maintaining  an  animal  in  nitrogen  equi- 
librium, and  recently  Osborne,  Mendel,  and  Ferry1  have  added 
glutenin  from  wheat  to  the  list  of  proteins  which  are  in- 
dividually capable  of  meeting  all  the  needs  of  an  animal  at 
least  so  far  as  maintenance  is  concerned,  although  up  to  the 
present  time  no  very  decided  positive  nitrogen  balances  have 
been  reported  in  experiments  where  the  animals  were  limited 
to  a single  protein  as  a source  of  nitrogen.  Zein,  gelatin  and 
others  have  been  found  not  to  possess  this  power. 

Prevailing  views  of  the  mechanism  of  protein  metabolism 
have  been  supported  and  developed  principally  by  Abder- 
halden.  The  amino  acids,  which  all  proteins  yield  on  hydroly- 
sis are  regarded  as  the  “ building  stones”  with  which  the  an- 
imal deals  in  constructing  its  specific  body  proteins.  It  is 
assumed  that  the  animal  has  no  synthetic  power  which  enables 
it  to  produce  from  other  complexes,  the  amino  acids  needed 
with  the  single  exception  of  glycocoll.  Henriques  2 has 
brought  forward  experimental  evidence  which  conflicts  with 
this  view  in  that  he  kept  rats  in  nitrogen  equilibrium  with  a 
ration  containing  no  protein  other  than  gliadin,  from  which 
lysin  is  absent.  Abderhalden3  has  disputed  the  possibility  of 
accomplishing  this  with  strictly  pure  gliadin. 

From  this  view  of  the  nature  of  protein  metabolism  it  fol- 
lows that  the  greater  the  similarity  of  the  molecule  of  food 
protein  to  that  of  the  specific  body  proteins,  the  greater  will 

1 Osborne,  Mendel,  and  Ferry,  Carnegie  Inst.  Bui.  156  (1911). 

2 Henriques,  Ztschr.  Physiol.  Chem.  60,  105  (1909). 

3 Abderhalden,  Ztschr.  Physiol.  Chem.  60,  425,  1909. 


Repair  Processes  of  Protein  Metabolism 


59 


be  the  food  value  to  the  animal.  It  also  follows  that  any  one 
of  the  essential  cleavage  products  which  is  present  in  smallest 
amount  in  food  protein,  determines  the  value  of  the  entire 
molecule  to  the  animal. 

The  most  elaborate  effort  to  test  the  validity  of  this  hypo- 
thesis is  the  work  of  Michaud4  who  says  “Mann  nur  dann  dem 
Eiweissminimum  am  nachsten  kommnt,  wenn  man  dass  Kor- 
pereigene  Eiweiss  verfiittert,  das  mann  sich  aber  umsomehr 
von  diesem  Minimum  entfernt,  als  mann  ein  in  seiner  chemis- 
chem  Konstit-ution  differentes  Eiweiss  gibt.”  If  this  is  strictly 
true,  a protein  wholly  lacking  in  one  or  more  cleavage  products 
found  in  the  tissues  of  an  animal  should  be  entirely  inadequate 
for  the  construction  of  new  body  protein  when  fed  alone. 
Gelatin,  gliadin  and  zein  are  in  this  class,  yet  all  investigations 
with  these  proteins  indicate  that  they  are  utilized  as  food  by 
the  animal  even  when  fed  as  the  sole  source  of  nitrogen.' 
When  fed  in  amounts  corresponding  to  the  fasting  level  of 
protein  metabolism  a considerable  portion  of  the  nitrogen  fails 
to  appear  in  the  urine  as  promptly  as  do  forms  known  not  to 
be  of  value  to  the  animal.  This  phase  of  the  subject  will  be 
further  treated  when  the  results  of  Michaud  are  examined  in 
some  detail. 

Michaud’s  method  would  appear  in  most  respects  to  be  a 
logical  one  for  determining  the  value  of  a particular  protein  to 
an  animal  for  purposes  of  repair  of  the  tissue  destroyed  in 
endogenous  metabolism.  The  plan  is  as  follows : The  animal 

is  reduced  to  its  lowest  possible  level  of  nitrogen  elimination 
by  feeding  a nitrogen-free  diet  (starch,  fat)  ; and  then  an 
amount  of  nitrogen  equivalent  to  the  lowest  daily  output  of 
which  the  animal  is  capable  is  fed  in  the  form  of  the  particular 
protein  to  be  studied.  The  degree  in  which  the  animal  utilizes 
the  protein  fed  in  replacing  the  tissue  daily  catabolized  is 
taken  as  a measure  of  its  value  as  a tissue-building  material. 
The  supposed  efficiency  of  the  method  is  based  on  the  assump- 
tion that  the  animal  will  utilize  the  nitrogen  presented  to  him 
for  repair  purposes  as  efficiently  as  it  is  possible  to  do  so. 


4 Michaud,  Ztschr.  Physiol.  Chem.  59,  405  and  421  (1909). 
s Compare  Merlin,  Amer.  Jour,  of  Physiol.  19,  285  and  20,  234 
(1907).  Also  Abderhalden,  Ztschr.  Physiol.  Chem.  60,  425  (1909); 
Henriques,  Ztschr.  Physiol.  Chem.  60,  105. 


GO  Wisconsin  Research  Bulletin  No.  21 

This  assumes  that  it  is  possible  for  an  animal  to  take  a given 
amount  of  nitrogen  and  to  convert  it  into  one  hundred  per 
cent  product,  i.  e.  body  tissue,  provided  its  eharcter  is  suit- 
able. This  assumption  would  seem  to  be  supported  by  the 
experimental  data  available.  Michaud  found  that  when  pro- 
tein equivalent  to  the  “protein  minimum”  was  fed  to  dogs  in 
the  form  of  casein,  or  dog  tissues,  when  the  animals  were  meta- 
bolizing at  their  minimum  level,  there  was  no  loss  of  nitrogen 
which  would  seem  to  indicate  a perfect  utilization  of  protein 
at  this  low  plane  of  intake. 

The  data  presented  in  this  paper  are  the  outgrowth  of  a series 
° ®^Perl“ents  undertaken  two  years  ago,  using  essentially  the 
method  of  Michaud,  to  compare  the  values  of  the  protein  mix- 
tures of  some  of  our  most  important  grains  as  nutrients  for  the 
pig.  It  was  hoped  that  specific  differences  would  be  shown 
by  this  method  between  grains  in  which  the  character  of  the 
protein  mixture  is  known  to  differ  so  markedly  as  in  the  wheat 
oat,  and  corn  kernels. 

Plan  of  Experiment 

The  animal  (pig)  was  fasted  for  two  or  three  days  with 
water  and  salts  until  it  would  take  a starch  solution  readily, 
and  then  it  was  given  fifty  calories  per  kilo  per  day  in  the  form 
of  starch  together  with  a salt  mixture  having  about  the  com- 
position of  the  ash  of  the  oat  kernel.  This  mixture  contained 
15.5%  K,0,  3.0%  Fe.A,  3.3%  CaO,  5.6%  MgO,  25.0%  P..O- : 
4.0%  S03,  and  44.2%  Cl. 

The  salts  and  starch  were  treated  with  a small  amount  of  boil- 
ing water  to  scald  a part  of  the  starch,  the  mass  quickly  stirred 
and  cool  water  added  in  amount  to  form  a soup  which  the  pig 
could  drink  readily.  This  ration  was  continued  with  daily 
quantitative  collections  of  the  urine  and  feces.  The  creatinin 
and  total  nitrogen  were  determined  daily.  From  the  amount 
of  nitrogen  eliminated  during  five  to  ten  days  in  the  form  of 
creatinin,  an  average  was  obtained  from  which  the  nitrogen  re- 
sulting from  endogenous  metabolism  was  calculated,  using  the 
formula  5.5  times  creatinin  nitrogen  equals  nitrogen  from  en- 
dogenous metabolism.  (See  page  78).  The  pig  was  then  main- 
tained on  the  nitrogen-free  diet  until  the  daily  output  of  nitro- 
gen in  the  urine  reached  about  this  level. 


Repair  Processes  of  Protein  Metabolism 


61 


When  the  urinary  nitrogen  was  entirely  of  endogenous  origin 
for  several  days,  as  shown  by  a nearly  constant  ratio  between 
the  nitrogen  as  creatinin  and  the  total  nitrogen  varying  be- 
tween 17 :100  and  19 :100,  the  pig  was  considered  to  be  ready 
for  an  experiment  in  nitrogen  feeding.  The  gram  to  be  stud- 
ied was  now  introduced  into  the  ration  and  an  isodynamic  quan- 
tity of  starch  withdrawn.  Since  feeding  a grain  always  leads 
to  an  increased  excretion  of  nitrogen  in  the  feces  it  was  as- 
sumed that  part  of  the  nitrogen  is  not  digested,  and  accord- 
ingly the  amount  of  grain  fed  was  such  that  if  90  per  cent  of 
the  nitrogen  were  digested  and  absorbed,  the  absorbed  part 
would  just  equal  the  amount  of  nitrogen  degraded  daily  in  en- 
dogenous metabolism.  Actually  the  increased  nitrogen  m the 
feces  amounted  to  somewhat  more  than  10  per  cent  of  the 
amount  fed  so  the  amount  absorbed  was  a little  less  than 
enough  to  meet  the  needs  of  the  pig  for  repairs. 

The  results  obtained  were  rather  surprising.  With  the  oat 
or  corn  kernel  there  was  no  appreciable  change  in  the  nitrogen 
content  of  the  urine  or  in  the  ratio  of  the  creatinin  nitrogen 
eliminated  to  the  total  nitrogen.  These  remained  essentially 
the  same*  as  in  the  latter  part  of  the  period  when  a nitrogen-free 
diet  was  given.  With  wheat  a small  rise  in  the  nitrogen  con- 
tent of  the  urine  was  observed  amounting  to  about  ten  to  fif- 
teen per  cent.  This  difference  was  entirely  too  small  to  ac- 
count for  well  known  differences  in  the  quantitative  relations 
among  the  cleavage  products  of  the  wheat  proteins  and  those 
of  animal  tissues  thus  far  studied.6  The  gluten  of  wheat  makes 
up  about  75  per  cent  of  the  total  protein  content  of  the  grain, 
and  yields  about  40  per  cent  of  its  nitrogen  as  glutaminic  acid, 
while  all  animal  proteins  thus  far  examined  contain  less  than 
17  per  cent  of  this  complex.  It  is  scarcely  possible  that  the 
mixture  of  cleavage  products  obtained  from  the  wheat  grain 
should  be  suitable  for  rearrangement  into  muscle  proteins  with 
an  efficient  utilization  of  nitrogen.  In  all  of  these  experiments 
with  individual  grains  the  efficiency  of  the  collection  of  the 
urine  was  tested  by  giving  one  or  more  doses  of  urea  to  see  if 


e Compare  the  papers  of  Osborne  and  his  co-workers  Amer.  Jour. 
Physiol.  22,  433,  23,  81  (1908),  24,  161  and  437  (1909).  Also  Ergeb. 
Physiol.  Abderhalden,  Ztschr.  Physiol.  Chem.  10,  47  (1910),  51,  311 
and  404  (1907). 


62 


Wisconsin  Research  Bulletin  No.  21 


the  nitrogen  administered  in  this  form  could  be  traced  into 
the  urine.  Urea  nitrogen  was  invariably  recovered  promptly 
to  the  extent  of  85  to  90  per  cent  in  the  urine  in  excess  of  the 
endogenous  nitrogen , and  such  excess  of  nitrogen  in  the  urine 
was  accompanied  by  a fall  of  the  per  cent  of  total  nitrogen  in 
the  form  of  creatinin.  These  results  led  me  to  undertake  a 
series  of  experiments  with  pigs  on  the  feeding  of: 

1.  Zein  nitrogen  equivalent  to  the  endogenous  urinary  nitro- 
gen; 

2.  Zein  nitrogen  equivalent  of  two  to  nine  times  the  endogen- 
ous nitrogen; 

3.  Gelatin  nitrogen  equivalent  to  the  endogenous  urinary 
nitrogen ; 

4.  Casein  nitrogen  equivalent  to  twelve  or  thirteen  times  the 
endogenous  urinary  nitrogen. 

The  plan  of  these  experiments  differed  from  those  of  Michaud 
in  certain  respects.  Michaud  took  as  the  “Eiweiss  Minimum” 
the  sum  of  the  urinary  and  fecal  nitrogen  excreted  by  the  ani- 
mal after  a long  period  on  a nitrogen-free  diet.  I have  taken 
the  nitrogen  of  the  urine  alone  as  an  index  to  the  amount  of 
nitrogen  to  be  fed,  and  have  interpreted  the  results  largely 
on  changes  in  the  nitrogen  content  of  the  urine  resulting  from 
feeding  the  forms  of  nitrogen  studied.  The  nitrogen  of  the 
feces  is  not  in  the  form  of  end  products  of  metabolism,  but 
represents  losses  which  might  be  termed  accidental  in  char- 
acter, such  as  cells  abraded  from  the  alimentary  tract,  the  res- 
idues of  the  secretions  which  escape  absorption,  together  with 
the  bacterial  growths  for  which  these  furnish  a medium,  as  well 
as  undigested  remains  of  feed.  These  are  all  sources  inde- 
pendent of  the  essential  tissue  metabolism . In  experiments  of 
this  character  we  should  deal  specifically  with  the  endogenous 
type  of  metabolism.  The  object  should  be  to  ascertain  the  in- 
fluence of  a particular  protein  on  the  repair  of  waste  of  an 
equivalent  amount  of  tissue  due  to  endogenous  metabolism  and 
not  to  attain  a state  of  nitrogen  equilibrium.  The  latter,  to 
attain  nitrogen  equilibrium,  would  require  enough  new  growth 
to  replace  those  losses  of  accidental  nature.  The  nitrogen  of 
the  feces  may  serve  as  an  approximate  indication  of  the  thor- 
oughness of  absorption  provided  adequate  records  are  obtained 
of  a fore  and  after  period  on  a nitrogen-free  diet. 


Repair  Processes  of  Protein  Metabolism 


63 


The  importance  of  taking  the  urinary  nitrogen  as  the  min- 
imum protein  metabolism  is  evident  because  of  the  vitiating  in- 
fluence of  feeding  nitrogen  at  too  high  a level  in  this  class  of 
work.  It  is  evident  that  if  we  are  to  measure  the  efficiency 
of  a particular  protein  by  the  excessive  excretion  of  nitrogen 
during  a feeding  period,  over  the  excretion  on  a nitrogen-free 
diet,  it  is  imperative  that  the  amount  of  nitrogen  fed  shall 
not  exceed  the  urinary  nitrogen  of  endogenous  origin.  If  any 
excess  over  this  amount  is  given,  the  animal  is  no  longer  limited 
by  the  one  essential  cleavage  product  present  in  smallest  quan- 
tity. He  is  given  some  choice  among  the  group  of  “building 
stones”  larger  than  is  necessary  for  repair. 

Michaud  makes  no  mention  of  giving  his  dogs  an  adequate 
supply  of  inorganic  salts.  In  some  of  his  feeding  periods  a cer- 
tain amount  of  salts  were  carried  by  the  protein  mixture  given, 
but  when  pure  proteins  were  fed  the  dogs  were  apparently  tak- 
ing a nearly  salt-free  ration.  In  my  own  experiments  a liberal 
supply  of  a salt  mixture  was  given  daily.  The  amount  of  water 
given  was  large  enough  to  keep  the  volume  of  urine  rather 
high.  It  was  believed  that  this  tended  to  a more  prompt,  uni- 
form and  complete  elimination  of  the  end  products  of  metabol- 
ism. 

The  investigation  of  this  subject  has  extended  over  two 
years,  and  a very  large  amount  of  data  has  been  collected. 
The  tables  here  presented  show  representative  protocols.  All 
the  data  collected  were  essentially  in  harmony.  The  pigs  used 
were  from  the  Wisconsin  Experiment  station  herd,  all  were  of 
the  larger  breeds,  and  were  of  the  best  growing  strains.  Ani- 
mals found  to  be  infested  with  intestinal  parasites  were  dis- 
carded. 

An  examination  of  Tables  I and  II  shows  that  when  a pig  has 
been  reduced  by  a long  continued  nitrogen-free  diet,  to  a condi- 
tion where  the  exogenous  type  of  metabolism  has  probably  en- 
tirely disappeared,  the  addition  of  an  amount  of  nitrogen  as 
zein  closely  approximating  that  derived  daily  as  end  products 
from  endogenous  metabolism,  the  rise  in  the  nitrogen  content 
of  the  urine  is  very  much  less  than  one  would  expect  for  a pro- 
tein of  this  character.  Although  three  amino  acids  are  lack- 
ing: glycocoll,  tryptophane  and  lysin,  all  of  general  occur- 

rence in  animal  tissues,  it  would  seem  evident  that  the  animal 


04 


Wisconsin  Research  Bulletin  No.  21 


lias  made  use  of  a large  part  of  the  nitrogen  of  this  protein. 
The  ratio  of  creatinin  nitrogen  to  total  nitrogen  falls  but  little 
from  what  it  is  on  a nitrogen-free  diet  which  shows  that  the 
nitrogen  of  the  urine  is  derived  principally  from  endogenous 
sources.  The  second  starch  period  eliminates  the  possibility 


TABLE  I.  FEEDING  ZEIN  EQUIVALENT  TO  ENDOGENOUS  METABOLISM 

Weight  at  beginning'  of  experiment  31  pounds  (16.82  kilograms). 

Pig  was  placed  in  the  cage  March  11,  and  fed  starch,  salt  mixture  and  water  durin; 
the  following  twenty  days.  No  record  of  the  nitrogen  output  until  March  26. 


Date 

Grams  N.  in 
food 

Gran 

March 
26 

Starch 

0 

.961 

27 

0 

.94 

28 

0 

.89 

29 

0 

.86 

30 

0 

.84! 

31 

0 

.96 

April 
1 

Starch,  zein 
.47 

1 

.95] 

2 

.95 

1.19) 

3 

.95 

.96! 

4 

.95 

1.10 

5 

.95 

1.08 

6 

.95 

1.13! 

7 

.95 

• 90  j 
1.24  | 

8 

.95 

9 

.47 

1.01 

10 

Starch 

0 

1 

1.001 

11.... 

0 

12 

0 

13 

0 

14 

0 

15 

Starch,  urea 
1.00 

16 

1.00 

17 

1.00 

18 

.50 

19 

Starch 

0 

20 

0 

21 

0 

22 

0 

Av.  .91  gr. 


Av.  1.07  gr. 


.83 

.61 

.81 

.77 

.80 

1.27 

1.52 
1.59 

1.21 

1.53 
1.23 


jrams  N. 
in 

feces 

Grams  N. 
as 

creatinin 

Per  cent  ol 
total  N.  as 
creatinin. 

.24 

.164 

17.08 

.24 

.143 

15.21 

.24 

.173 

19.05 

.24 

.165 

19.18 

.24 

.143 

17.02 

.24 

.154 

15.10 

.30 

.166 

17.46 

.30 

.149 

12.52 

.30 

.144 

15.00 

.30 

.143 

13.00 

.30 

.168 

15.37 

.30 

.141 

12.47 

.30 

.139 

15.44 

.30 

.185 

14.92 

.30 

.157 

15.54 

.28 

.157 

15.70 

.28 

.154 

18.55 

.28 

.122 

19.99 

.28 

.133 

16.42 

.28 

.165 

21.42 

.28 

.147 

18.37 

.28 

.157 

12.36 

.28 

.132 

8.66 

.28 

.156 

9.81 

.28 

.162 

13.39 

.28 

.151 

9.86 

.28 

.141 

11.47 

.28 

.160 

17.97 

of  a lag  in  the  excretion  of  the  zein  nitrogen.  The  urea  period 
following  the  second  starch  period  shows  how  quickly  the  com- 
position of  the  urine  changes  when  a form  of  nitrogen  useless 
to  the  animal  is  introduced.  The  total  nitrogen  rises  at  once 
and  with  this  goes  a marked  change  in  the  per  cent  of  the 
total  nitrogen  in  the  form  of  creatinin.  Reference  will  he  made 
later  to  the  influence  of  similar  amounts  of  gelatin  nitrogen 
when  administered  to  an  animal  under  these  conditions. 

It  has  been  customary  with  many  workers  in  interpreting 
the  new  data  in  experiments  for  determining  the  nutritive  value 
of  proteins,  to  judge  from  a plus  or  minus  nitrogen  balance  as 


Repair  Processes  of  Protein  Metabolism 


65 


to  whether  the  protein  in  question  is  sufficient  or  insufficient  to 
maintain  the  animal  in  nitrogen  equilibrium.  "We  should  go 
farther  than  this.  Looked  at  in  this  way  the  protocols  shown 
in  Tables  I and  II  both  show  decided  nitrogen  deficits.  If  the 
nitrogen  content  of  the  feces  is  enough  greater  in  the  period 


TABLE  II.  FEEDING  ZEIN  EQUIVALENT  TO  ENDOGENOUS  METABOLISM 

Weight  of  pig  at  beginning  of  experiment  158  pounds  (71.8  kilograms). 

December  11  to  17  fed  50  calories  per  kilo  as  starch,  with  salt  mixture  and  water. 

Urine  not  examined. 

During  the  four  days’  fast  the  animal  was  given  two  grams  of  agar-agar  daily  to 
insure  regular  evacuations.  The  feces  were  collected  from  December  14  to  21  inclu- 
sive, no  agar-agar  being  given.  The  feces  for  December  21  were  marked  off  by  a 
fifteen-gram  dose  Ca3  (POi)2.  The  nitrogen  content  for  this  period  was  6.16  grams 
or  .77  grams  per  day.  The  remainder  of  the  protocol  is  given  in  tabular  form. 


Date 

Grams  N.  in 
food 

Grams  N.  in 
urine 

Grams  N.  in 
feces 

Grams  N.  as 
creatinin 

Per  cent,  of 
N.  as  crea- 
tinin 

December. 
18 

Starch 

0 

2.59 

.77 

.48 

18.64 

19 

0 

2.61 

.77 

.44 

17.01 

20 

0 

2.66 

.77 

.50 

18.94 

21 

0 

2.58 

.77 

.48 

18.83 

22 

Starch. zein 
2.62 

2.98 

.72 

.45 

15.10 

23 

2.62 

2.48 

.72 

.49 

18.77 

24 

2.62 

2.75 

.72 

.47 

17.09 

25 

2.62 

2.97 

.72 

.52 

17.51 

26 

2.62 

2.62 

.72 

.48 

18.32 

27 

2.62 

2.56 

.72 

.39 

15  23 

28 

2.62 

3.87 

.72 

.70 

18.09 

29 

2.62 

3.11 

.72 

.48 

15.43 

30 

2.62 

3.85 

.72 

.55 

14.28 

31 

2.62 

3.07 

.72 

.48 

15.63 

January  . . 
1 

1.31 

2.37 

.72 

.19 

16.45 

2 

Starch 

0 

3.02 

.72 

.46 

15.20 

3 

0 

2.43 

.68 

.52 

21.40 

4 

0 

2.94 

.68 

.46 

15.64 

5 

0 

2.67 

.68 

.48 

17.97 

6 

Starch,  urea 
2.62 

2.58 

.68 

.48 

18.60 

7 

2.62 

4.90 

.68 

.48 

9.79 

8 

0 

5.04 

.68 

.52 

10.31 

9 

0 

3.01 

.68 

.47 

15.61 

10 

0 

2.70 

.68 

.48 

17.40 

11 

0 

2.66 

.68 

.48 

18.04 

when  the  protein  is  fed,  than  it  was  during  the  last  portion  of 
the  nitrogen-free  period  to  indicate  with  certainty  an  incom- 
plete digestion  and  absorption  of  the  food  protein,  the  increase 
in  the  fecal  nitrogen  should  be  considered  as  an  index  to<  the  de- 
gree of  absorption.  But  knowing  the  amount  of  nitrogen 
absorbed,  our  judgment  concerning  its  value  for  replacing  the 
endogenous  loss  should  be  based  entirely  on  changes  observed 
in  the  urine  viz.,  on  the  increase  if  any,  in  the  total  nitrogen, 
and  the  change  in  the  ratio  between  the  nitrogen  as  creatinin 
and  the  total  nitrogen.  Looked  at  from  this  standpoint,  the 


66 


Wisconsin  Research  Bulletin  No.  21 


tables  give  an  accurate  idea  of  the  nutritive  value  of  the  nitro- 
gen of  zein  for  repair  purposes. 

During  the  nine  days  when  zein  was  fed  (Table  I)  the  pig 
would  have  lost  8.19  grams  (9x.91)  of  nitrogen  if  he  had  been 
kept  on  a starch  diet.  He  was  fed  7.59  gramis  of  nitrogen  as 
zein  and  his  total  output  of  nitrogen  in  the  urine  during  these 
nine  days  was  9.61  grams  which  is  only  .16  gram  more  per  day 
than  he  would  have  excreted  if  no  nitrogen  had  been  given. 
The  deficit  of  urinary  nitrogen  for  the  nine  days  was  only  1.4 
grams.  It  would  seem  therefore  that  the  7.59  grams  of  nitro- 
gen fed  had  replaced  6.77  grams  of  this  element  lost  through 
endogenous  metabolism  in  this  period. 

An  inspection  of  Table  II  shows  that  during  the  eleven  days 
of  zein  feeding  the  output  of  nitrogen  in  the  urine  was  32.6 
grams,  whereas  the  nitrogen  output  on  a starch  diet  as  calcu- 
lated from  the  creatinin  output  during  the  same  time  as  ob- 
served in  the  preliminary  starch  period  would  have  been  28.8 
grams.  During  this  period  27.5  grams  of  nitrogen  were  fed 
as  zein.  The  loss  of  nitrogen  by  the  pig  was  thereby  reduced 
from  28.8  to  3.8  grams  during  the  eleven  days.  Since,  how- 
ever, in  the  starch  period  following,  .58  grams  of  nitrogen 
was  excreted  in  excess  over  what  was  to  be  expected,  the  loss 
should  be  considered  about  4.5  grams.  It  would  appear  that, 
although  zein  is  lacking  in  three  amino  acids,  its  efficiency 
as  a repair  material  in  cellular  metabolism  is  quite  high. 

The  creatinin  output  during  the  first  ten  days  on  a starch 
diet  averaged  .201  gram  of  nitrogen  per  day.  From  this  the 
nitrogen  equivalent  to  the  endogenous  metabolism  of  the  ani- 
mal was  calculated  to  be  1.105  grams.  (.201x5.5).  The  total 
nitrogen  in  the  urine  was  not  determined  during  the  first  five 
days.  The  nitrogen  of  the  urine  had  not  fallen  to  the  lowest 
possible  level  at  the  end  of  ten  days  on  a nitrogen-free  diet,  as 
shown  by  the  value  of  the  endogenous  metabolism  calculated 
from  the  creatinin  output,  and  also  by  the  fact  that  the  nitrogen 
appearing  as  creatinin  was  still  only  about  16  per  cent  of  the 
total.  It  was  assumed,  however,  that  the  pig  was  sufficiently 
freed  from  the  end  products  of  metabolism  for  the  purpose  of 
this  experiment  which  was  to  determine  whether  an  appre- 
ciable retention  of  nitrogen  could  be  induced  by  feeding  zein 
in  excess  of  the  “ maintenance  ’’  requirements  of  the  animal. 


Repair  Processes  of  Protein  Metabolism 


67 


All  previous  efforts  to  induce  growth  in  animals  by  feeding 
zein  as  the  sole  source  of  protein  have  failed  entirely7  but  as 
will  appear  later  in  this  paper  the  pig  appears  to  be  exception- 


TABLE  III.  FEEDING  ZEIN  ABOVE  NEEDS  FOR  REPAIR 

Record  of  a pig  on  a ration  supplying  nitrogen  in  excess  of  the  needs  of  the  animal 
for  repair,  in  which  zein  was  the  only  source  of  protein.  Weight  of  pig  at  beginning 
of  experiment  63.5  pounds  (28.80  kilograms).  Weight  at  end  of  experiment  64.5  pounds 
(29.32  kilograms). 


Date 

Grams  N.  in 
food 

Grams  N.  in 
urine 

Grams  N.  in 
feces 

Grams  N.  in 
urine  as 
creatinin 

June 

Starch 

29 

0 

1.78 

.59 

.201 

30 

0 

1.70 

.59 

.189 

July 

1 

0 

1.42 

.59 

.194 

2 

0 

1.34 

.59 

.223 

3 

0 

1.29 

.59 

.210 

Starch,  zein 

4 

1.78 

1.20 

.57 

.204 

5 

1.78 

1.40 

.57 

.200 

6 

1.78 

2.19 

.57 

.197 

7 

1.78 

1.83 

.57 

.194 

8 

1.78 

1.73 

.57 

.174 

9 

1.78 

1.79 

.57 

.174 

10 

1.78 

1.95 

.57 

.214 

11 

1.78 

1.99 

.57 

.189 

12 

1.78 

2.13 

.57 

.199 

13 

1.78 

2.13 

.57 

.195 

14 

2.23 

2.04 

.61 

.214 

15 

2.23 

2.25 

.61 

.206 

16 

2.23 

3.23 

.61 

.185 

17 

2.23 

1.08 

.61 

.194 

18 

2.23 

2.43 

.61 

.192 

19 

2.23 

2.52 

.61 

.234 

20 

2.23 

2.38 

.61 

.201 

21 

3.35 

2.16 

.66 

.204 

22 

3.35 

2.80 

.66 

.182 

23 

3.35 

2.16 

.66 

.182 

24 

3.35 

3.78 

.66 

.195 

25 

3.35 

2.89 

.66 

.214 

26 

3.35 

2.98 

.66 

.200 

27 

3.35 

2.82 

.66 

.170 

28 

3.35 

3.21 

.66 

.196 

29 

3.35 

2.95 

.66 

.209 

30 

3.35 

3.14 

.66 

.228 

31 

0 

2.96 

.66 

.199 

August 

Starch 

1 

0 

2.64 

.66 

.204 

2 

0 

2.64 

.66 

.210 

3 

0 

1.61 

.66 

4 

0 

1.62 

.66 

5 

0 

1.08 

.66 

6 

0 

1.29 

.66 

ally  efficient  in  the  utilization  of  food-stuffs  so  it  appeared  pos- 
sible that  more  favorable  results  might  be  met  in  the  case  of 
zein.  The  record  given  in  Table  III  begins  with  the  sixth 
starch  day. 

Table  III  shows  the  behavior  of  a pig  on  a ration  supplying 
zein  in  quantities  a high  as  three  times  greater  than  the  endog- 

7 Willcock  and  Hopkins,  Jour.  Physiol.  35,  117  (1906-7).  Henriques, 
Ztschr.  Physiol.  Chem.  60,  425  (1909). 


68 


Wisconsin  Research  Bulletin  No.  21 


enous  metabolism.  If  we  consider  the  record  beginning  July 
4 and  ending  with  the  experiment  (34  days),  the  nitrogen 
fed  was  66.9  grams.  The  total  elimination  in  the  urine  of 
the  entire  period  was  76.7  grams  of  nitrogen  leaving  a neg- 
ative balance  of  9.8  grams  of  nitrogen  in  the  urine.  The 
nitrogen  content  of  the  feces  in  the  periods  separated  shows 
that  the  digestion  was  complete.  The  after  period  on  starch 
was  made  long  enough  to  bring  the  pig  to  the  same  level  of 
nitrogen  elimination  as  at  the  beginning  of  the  feeding  of 
zein.  We  must  consider,  however,  that  if  the  pig  had  received 
no  protein  during  these  34  days  (starch  diet)  the  nitrogen 
elimination  in  the  urine  would  have  been  about  37.4  grams. 
By  feeding  zein  the  loss  of  nitrogen  from  endogenous  metabol- 
ism was  reduced  by  27.6  grams.  That  is  27.6  grams  of  this 
element  degraded  through  tissue  catabolism  was  repaired 
from  the  zein  fed.  Of  the  excessive  nitrogen  fed,  however, 
none  was  stored  as  gain.  The  same  care  was  given  to  the 
collection  of  the  excreta  as  in  the  case  of  other  animals  in 
experiments  in  which  the  addition  of  a small  amount  of  urea 
to  the  diet  led  to  a prompt  detection  of  a corresponding  rise 
in  the  nitrogen  excreted.  The  above  difference  is  too  large  to 
be  charged  to  experimental  error. 

It  might  be  urged  that  the  amount  of  zein  fed  in  this  case 
was  too  small  in  excess  of  the  maintenance  needs  to  be  con- 
ducive to  growth.  It  is  true,  we  know  almost  nothing  of  the 
influence  of  the  plane  of  protein  intake  on  the  tendency  of 
the  growing  animal  to  construct  new  body  protein.  Table 
IV  is  therefore  presented  giving  the  record  of  a pig  which 
took  a relatively  high  protein  diet  in  which  zein  furnished  all 
the  nitrogen. 

The  pig  from  which  the  data  given  in  Table  IY  were  ob- 
tained weighed  51  pounds  (23.18  kilograms)  at  the  beginning 
and  lost  one  pound  during  the  experiment.  He  was  fed  a 
starch  and  salt  diet  (50  calories)  during  an  eleven  day  fore 
period,  during  which  the  average  nitrogen  output  in  the  form 
of  creatinin  was  .19  grams  daily.  Assuming  that  he  would 
finally  have  established  a ratio  of  18.5 :100  between  creatinin 
nitrogen  and  total  nitrogen,  the  endogenous  metabolism  of 
this  pig  was  1.04  grams  of  nitrogen  daily.  This  level  was  not 
reached  in  eleven  days,  the  nitrogen  content  of  the  urine  on 


Repair  Processes  of  Protein  Metabolism 


69 


the  tenth  and  eleventh  days  of  the  fore  period  being  1.37  and 
1.41  grams  respectively.  However,  it  was  believed  that  the 
animal  was  in  a satisfactory  condition  for  an  experiment  of 
this  character.  He  was  thereafter  fed  zein  as  indicated  in 

Table  IV. 

TABLE  IV.  NITROGEN  EXCRETION  FROM  HIGH  INTAKE  OP  ZEIN 


Date 

Grams 
N.in  food 

Grams 
N.in  urine 

Grams 
N.in  feces 

i 

February 

4.47 

1.41 

.74 

4.47 

2.35 

.74 

4.47 

3.27 

.74 

6.59 

4.35 

.74 

5 ... 

8.82 

4.23 

.74 

9.24 

8.46 

.74 

9.53 

8.28 

.74 

8 

9.53 

8.44 

.74 

u 

9.53 

8.13 

.74 

in 

9.53 

8.23 

,.74 

11  

9.53 

8.18 

.74 

9.53 

7.43 

.74 

13  

9.53 

8.23 

.74 

14  ..  

9.53 

8.55 

.74 

15  

9.53 

8.60 

.74 

9.53 

7.96 

.74 

17 

9.53 

8.60 

.74 

18  

Starch 

0. 

8.62 

.74 

19  

0. 

6.04 

.74 

20 

0. 

4.78 

.74 

21 

0. 

3.70 

.74 

22  

0. 

2.34 

.64 

23 

0. 

2.25 

.64 

24  

0. 

1.45 

.64 

25  

0. 

1.45 

.64 

•>«  

0. 

1.26 

.64 

Total 

142.89 

146.59 

18.74 

The  record  in  Table  IV  shows  a marked  utilization  of  zein 
nitrogen  in  supplying  material  for  the  replacement  of  nitrogen 
lost  through  endogenous  metabolism.  Although  the  table  as 
a whole  shows  a negative  balance,  it  is  by  no  means  great 
enough  to  indicate  a total  lack  of  usefulness  of  zein  nitrogen. 

During  the  first  seventeen  days  of  the  experiment  the  pig 
was  fed  142.28  grams  of  nitrogen.  On  account  of  the  lag  in 
the  nitrogen  excretion  it  is  necessary  to  consider  the  feeding 
period  and  the  starch  period  following,  together.  During  the 
twenty-six  days  covered  by  these  periods  the  nitrogen  excreted 
in  the  urine  was  150.59  grams.  The  total  deficit  was,  there- 
fore 8.31  grams.  If  the  pig  had  been  kept  on  a starch  diet 
he  would,  judging  from  the  creatinin  output,  have  lost  27.04 
grams  of  nitrogen  from  endogenous  metabolism1.  If  none  of 
the  zein  had  been  utilized  by  the  pig  he  should  have  excreted 


70 


Wisconsin  Research  Bulletin  No.  21 


in  his  urine  all  of  the  nitrogen  taken  in  this  form  together 
with  the  27.04  grams  derived  from  endogenous  sources.  The 
nitrogen  elimination  in  the  urine  should,  under  these  circum- 
stances have  been  142.28  -f-  27.04  ==  169.32  grams  for  the  twenty- 
six  days.  The  difference  between  this  figure  and  the  nitrogen 
found  in  the  urine  (169.32 — 150.59=18.73)  we  are  to  suppose 
was  replaced  from  the  zein  fed.  No  new  growth  has  been 
demonstrated  in  any  case  when  zein  has  been  the  only  protein 
supplied.  These  results  with  zein  were  so  unexpected  in  char- 
acter that  it  was  thought  desirable  to  make  similar  experi- 
ments with  gelatin,  using  the  pig,  in  order  to  compare  the 
efficiency  of  this  species,  with  others  in  the  utilization  of  this 
form  of  nitrogen.  Gelatin  has  been  used  in  nutrition  studies 
more  than  any  of  the  other  “ incomplete”  proteins.  In  most 
of  the  recorded  experiments  it  has  seemed  doubtful  whether 
the  animals  were  fed  gelatin  nitrogen  at  the  same  level  as 
the  endogenous  metabolism,  and  that  this  fact  would  account 
for  the  lack  of  uniformity  in  the  results  of  various  observers, 
as  to  the  nutritive  value  of  this  substance.  Accordingly  six 
experiments  were  carried  out  on  the  same  plan  as  used  in  the 
experiments  recorded  in  Tables  I and  II.  For  presentation 
here,  the  record  is  selected  of  a pig  fed  starch  during  a long 
fore  period,  then  zein,  followed  by  a starch  period,  then  gela- 
tin at  the  sarnie  level  as  zein,  and  lastly  a starch  period  long 
enough  to  allow  the  pig  to  return  to  his  endogenous  level. 
(See  Table  V.)  The  results  were  all  in  harmony  and  showed 
an  apparent  utilization  of  between  50  and  60  per  cent  of  the 
nitrogen  given,  in  this  form  for  purposes  of  repair.  The  util- 
ization of  gelatin  nitrogen  fell  in  all  cases  far  short  of  that 
of  zein.  Not  more  than  20  per  cent  of  the  nitrogen  fed  as 
zein  has  ever  been  traced  into  the  urine  in  excess  of  the  en- 
dogenous output,  when  the  amount  fed  did  not  exceed  5.5 
times  the  nitrogen  daily  eliminated  by  the  animal  as  creatinin. 
When  gelatin  was  fed  in  like  amounts,  between  40  and  50 
per  cent  of  the  nitrogen  given  was  promptly  recovered  in  the 
urine  in  excess  of  what  should  have  been  found  had  a nitro- 
gen-free diet  been  taken  during  the  same  period. 

When  considering  experiments  like  that  shown  in  Table 
IV,  we  are  confronted  by  the  fact  that  no  very  decided  posi- 
tive nitrogen  balance  has  as  yet  been  obtained  in  experiments 


Repair  Processes  of  Protein  Metabolism 


71 


where  but  one  protein  has  been  given.  I have  reported  ex- 
periments in  which  an  appreciable  growth  was  secured  in 
young  rats  fed  a mixture  of  two  proteins,  together  with  carbo- 
hydrates, fats  and  the  necessary  inorganic  salts.8  These  are 
the  most  successful  of  any  yet  reported,  with  so  simple  a ra- 

TABLE  V.  ZEIN  AND  GELATIN  EQUIVALENT  TO  ENDOGENOUS  METABOLISM 


Weight  of  pig  150.5  pounds  at  beginning:  152  pounds  at  end  of  experiment. 

The  pig  was  fed  starch  (90  calories  per  kilogram),  a salt  mixture,  and  water  during 
a twenty-four  day  period.  A Quantitative  record  was  kept  daily  of  the  nitrogen 
content  of  the  urine,  and  feces  and  also  of  the  creatinin  output.  The  record  pre- 
sented begins  with  the  twentieth  day  on  a nitrogen-free  diet. 


Date 

Grams  N.  in 
food 

Grams  N.  in 
, urine 

Grams  N. 
in  feces 

Grams  N. 
as 

creatinin 

Per  cent  of 
total  N.  as 
creatinin 

Dec. 

Starch 

17 

0 

2.54 

.94 

.476 

18.74 

18 

0 

2.59 

.94 

.485 

18.72 

19 

0 

2.64 

.94 

.474 

18.16 

20 

0 

2,92 

.94 

.465 

15.92 

21 

0 

2.59 

.94 

.475 

18.34 

Starch  zein 

22 

2.62 

1.98 

1.06 

.325 

16.40 

23 

2.62 

2.48 

1.06 

.516 

20.80 

24 

2.62 

2.75 

1.06 

.574 

20.87 

25 

2.62 

2.98 

1.06 

.425 

14.26 

26 

2.62 

2.G2 

1.06 

.465 

17.74 

27 

2.62 

2.56 

1.66 

.473 

18.47 

28 

2.62 

3.87 

1.06 

.494 

12.78 

29 

2.62 

3.11 

1.06 

.481 

15.46 

30 

2.62 

3.85 

1.06 

.476 

12.36 

Starch 

31 

0 

3.07 

1.06 

.472 

12.36 

Jan. 

1 

0 

2.37 

1.06 

.473 

19.95 

2 

0 

3.69 

1.06 

3 

0 

2.43 

1.06 

.481 

19.79 

4 

0 

2.94 

1.06 

.471 

16.00 

Starch  gelatin. 

5 

2.62 

3.02 

1.06 

.474 

15.69 

6 

2.62 

3.15 

1.04 

.465 

14.73 

7 

2.62 

3.78 

1.04 

.473 

12.51 

8 

2.62 

4.32 

1.04 

.475 

10.99 

9 

2.62 

3.20 

1.04 

.472 

14.75 

10 

2.62 

5.57 

1.04 

.492 

8.83 

11 

2.62 

3.46 

1.04 

.476 

13.75 

12 

2.62 

3.24 

1.04 

.474 

14.62 

Starch 

13 

0 

3.02 

1.04 

14 

0 

3.35 

1.04 

15 

0 

2.60 

1.04 

16.. 

0 

3.00 

1.04 

17 

0 

2.40 

1.04 

tion.  The  growth  was  in  no  case  comparable  to  normal  growth 
in  the  rat  and  the  high  degree  of  uncertainty,  and  general 
unsatisfactoriness  of  the  results,  render  this  line  of  experi- 
mentation very  discouraging  as  a means  of  throwing  light  on 
the  chemical  processes  of  metabolism.  However,  the  impor- 


s Amer.  Jour.  Physiol.  25,  120  (1909). 


72 


Wisconsin  Research  Bulletin  No.  21 


tance  of  finding  a mixture  of  pure  chemical  substances  on 
which  an  animal  will  grow  in  a nearly  normal  way  is  great  and 
warrants  the  expenditure  of  much  effort  in  this  direction. 

In  feeding  rations  compounded  of  relatively  pure  substances 
my  attention  has  been  repeatedly  attracted  by  the  marked 
differences  exhibited  by  individuals  of  the  same  species,  in 
their  behavior  toward  such  mixtures.  Osborne  and  Mendel 
have  called  attention  to  the  same  thing  in  their  work.0  This 
fact  together  with  a remarkable  difference  in  the  attitude  of 
animals  of  different  species  toward  rations  made  up  of  natur- 
ally occurring  mixtures  but  derived  from  restricted  sources, 
has  been  strongly  emphasized  in  my  own  experience.  Of  twenty- 
four  young  rats  ranging  from  30  to  70  grams  in  weight,  when  fed 
on  wheat  alone,  but  nine  made  gains  of  15  grams  or  more  during 
the  first  three  months.  Eleven  made  no  gains  at  all,  but  died 
in  forty  to  sixty  days  having  suffered  no  appreciable  change 
in  weight  during  that  time.  Only  two  doubled  their  weight 
on  this  ration.  All  of  these  rats  if  fed  a normal  ration  should 
have  attained  weights  of  from  190  to  225  grams  in  this  period. 
This  happened,  not  in  a single  experiment,  but  in  three  ex- 
periments in  three  different  years.  In  a similar  trial  with 
three  young  pigs  of  fifty  pounds  weight,  in  which  the  wheat 
kernel  was  the  only  food  given,  and  the  animals  were  kept 
away  from  vegetation,  almost  normal  growth  was  observed 
during  the  first  six  months,  and  in  the  case  of  one  animal  with 
which  the  ration  was  continued  nine  months  a weight  of  190 
pounds  was  attained.  This  observation  together  with  the 
knowledge  that  a pig  will  readily  take  a sufficient  quantity 
of  a starch  solution  to  cover  his  energy  requirements,  and  will 
continue  to  do  so  for  at  least  thirty-five  days  with  no  evidence 
of  anorexia  and  no  appreciable  loss  in  weight  led  me  to  try 
the  pig  on  a mixture  of  casein,  starch,  salts  and  water.  The  re- 
sults were  gratifying  and  have  convinced  me  that  the  pig  is 
particularly  suitable  for  this  class  of  work.  The  records  of  two 
pigs  kept  in  metabolism  cages  and  fed  this  simple  mixture,  are 
shown  in  Tables  VI  and  VII.  It  will  be  seen  on  inspecting 
Table  VI  that  a pig  of  48  pounds  began  to  retain  nitrogen  at 
a remarkable  rate  and  continued  to  do  so  at  a perfectly  satis- 


o Osborne  and  Mendel,  Carnegie  Inst.  Bui.  156  (1911). 


Repair  Processes  op  Protein  Metabolism 


73 


factory  rate  during  a period  of  thirty-six  days.  On  February 
13  it  was  noticed  that  the  pig  was  not  as  eager  to  be  let  into 
the  feeding  stall  as  he  usually  was,  but  he  entered  of  his  own 
accord  and  ate  all  of  his  food.  From  that  time  on  he  grew 

TABLE  VI.  FEEDING  CASEIN  AS  THE  ONLY  SOURCE  OF  PROTEIN 

Weight  of  pig  48  pounds  (21.81  kilograms)  at  beginning,  55  pounds  (25  kilograms)  a 
end  of  experiment.  The  pig  was  allowed  to  go  two  days  with  water  alone,  and  was 
then  fed  100  calories  per  kilogram  as  starch,  an  inorganic  salt  mixture  with 
additional  ground  rock  phpsphate  and  casein  as  indicated  in  the  table. 


Date 

Grams  N. 
in  food 

Grams  N. 
in  urine 

Grams 
N.  in 
feces 

Total 
grams  of 
N.  output 

Grams 
N.  as 
creatinin 

Jan. 

13 

14.15 

2.85 

.60 

3.45 

.230 

14 

14.15 

3.00 

.60 

3.60 

.266 

15 

14.15 

5.86 

.60 

6.46 

.247 

16 

14.15 

5.31 

.60 

5.91 

.234 

17 

14.15 

5.75 

.60 

6.35 

.236 

18 

14*15 

5.07 

.60 

5.67 

.266 

19 

14.15 

5.09 

.60 

5.69 

.271 

20 

14.15 

5.53 

.60 

6.13 

.243 

21 

14.15 

5.51 

.60 

6.11 

.258 

22  

14.15 

5.79 

.60 

6.39 

.2/2 

23 

14.15 

5.27 

.60 

5.87 

.291 

24 

14.15 

5.83 

.60 

6.43 

.243 

25 

14.15 

4.70 

.60 

5.30 

.278 

14.15 

6.11 

.60 

6.71 

.279 

27 

14.15 

6.39 

.60 

6.99 

.284 

28 

14.15 

6.39 

.60 

6.99 

.293 

20 

14.15 

6.09 

.60 

6.69 

.248 

30 

14.15 

6.17 

.60 

6.77 

.289 

31 

14.15 

6.43 

.60 

7.03 

.286 

Feb. 

14.15 

5.90 

.60 

6.50 

.293 

2 

3  

4 

7.10 

7.93 

.60 

8.53 

.324 

7.10 

8.70 

.60 

9.30 

.285 

10.66 

9.04 

.60 

9.64 

.277 

5 

14.21 

7.67 

.60 

8.27 

.320 

14.21 

7.50 

.74 

8.24 

.302 

7 

8.. 

q 

14.21 

9.00 

.74 

9.74 

.284 

14.21 

8.04 

.74 

8.78 

.287 

14.21 

8.39 

.74 

9.13 

.325 

in 

14.21 

8.31 

.74 

9.05 

.290 

11 

14.21 

8.16 

.74 

8.90 

.311 

12 

14.21 

7.77 

.74 

8.51 

.328 

13 

14.21 

9.57 

.74 

10.31 

.307 

14 

14.21 

•9.66 

.74 

10.40 

.272 

15 

14.21 

9.92 

.74 

10.66 

.330 

in 

14.21 

9.75 

.74 

10.49 

.355 

17 

9.82 

74 

10.56 

.312 

Total . . 

478.38 

248.27 

23.28 

271.55 

The  pig  died  February  17. 

Nitrogen  retained  in  the  thirty-six  days 

Average  creatinin  N.  during  the  first  five  days. 
Average  creatinin  N.  during  the  last  five  days. 


206.83  grams 
.242  grams 
.315  grams 


Increase 


.073  grams 


more  listless,  but  his  appetite  remained  good  and  he  ate  all  of 
the  food.  The  nitrogen  output  had  been  gradually  increasing 
after  the  first  two  weeks.  On  the  evening  of  February  16, 
when  he  started  to  the  feeding  stall  to  eat,  he  fell  and  could 


74 


Wisconsin  Research  Bulletin  No.  21 


not  get  up.  He  seemed  to  have  lost  the  use  of  his  legs,  espe- 
cially the  hind  legs.  He  was  evidently  in  great  pain  for  he 
squealed  loudly  and  trembled  all  over.  The  muscles  were  re- 
laxed during  this  attack.  After  a quarter  of  an  hour  the  evi- 


TABLE  VII.  FEEDING  CASEIN  AS  THE  ONLY  SOURCE  OF  PROTEIN 
Ration:  casein  starch,  ash  of  milk,  NaCl  and  water. 

Initial  weight  43.5  pounds;  final  weight  51  pounds. 

Th)fiff».WaS  kept.?n.  a starch  diet  (N-free)  from  March  11  to  April  16 
0 dict  was  ■,9‘ ffram  Ni 
Began  feeding  casein  April  16. 


Date 

Grams  N.  in 
food 

Grams  N.  in 
urine 

Grams  N.  ir 
feces 

i Grams  total 
N. output 

Grams  N.  as 
creatinin 

April 

4 

5 

6 

7 

8 
9 

10 

11 

12 

13 

14 

15 

Starch 

0. 

0. 

0. 

0. 

0. 

0. 

0. 

0. 

0. 

0. 

0. 

0. 

1.64 

.76 

1.24 

1.07 

1.12 

1.01 

1.12 

1.10 

1.02 

1.21 

1.02 

.91 

.34 

.34 

.34 

.34 

.34 

.34 

.34 

.34 

.34 

.34 

.34 

.34 

1.98 

1.10 

1.58 

1.41 

1.46 

1.35 
1.46 
1.44 

1.36 
1.55 
1.36 
1.25 

.204 

.188 

.174 

.200 

.189 

.205 

.193 

.207 

.184 

.191 

.192 

16 

17 

18 

19 

20 
21 
22 

23 

24 

25 

26 

27 

28 

Casein,  starch 

Li 

.39 

.39 

u . oO 

13.72 

iq  79 

.99 

2.22 

2.67 

5.85 

4.14 

4.77 

4.20 

5.28 
5.41 

4.29 
11.33 

9.46 

1.38 

2.61 

ID  • ( 6 
IQ  70 

.39 

3.06 

ID.  { 6 
IQ  79 

.39 

6.24 

ID.  (6 
IQ  79 

.39 

4.53 

±0.(6 
IQ  79 

.39 

5.16 

ID . i 6 
A GO 

.39 

4.59 

O.oO 

13.72 

6.86 

13.72 

10.97 

in  07 

.39 

.39 

.39 

.39 

.39 

5.67 
5.80 

4.68 
11.72 
9.85 

.219 

.203 

.234 

.212 

29 

30 

May 

2 

10  Q7 

11.01 

8.34 

.39 

11.40 

iu.y/ 

1 O 07 

.39 

8.73 

lu.y* 

6.53 

.39 

6.92 

10.97 

lO  07 

5.66 

3.63 

.39 

6.05 

3 

lu.yi 
1 0 07 

.39 

4.02 

4 

lu.y/ 

in  07 

5.53 

.39 

5.92 

5 

6 
7 

iu.y< 

10.97 

.0 

r A Q 

4.19 

3.41 

0. 

.39 

.39 

.39 

4.58 

3.80 

.39 

.246 

.251 

8 

9 

D.4o 

8.22 

16.97 

5.43 

3.38 

8.11 

.39 

.39 

.39 

5.82 

3.77 

8.50 

.367 

.253 

.262 

Total  . 

259.74 

139.05 

13.44 

152.49 

ASM 


dence  of  pain  disappeared,  but  the  animal  was  still  almost  help- 
less. When  helped  to  the  trough  he  ate  all  of  his  food  and  was 
helped  back  again  to  the  cage.  The  next  day  he  seemed  to  rest 
easily  and  ate  a portion  of  his  food  when  assisted  to  the  trough. 
When  left  alone,  he  lay  flat  on  his  side  with  the  legs  extended 
but  not  rigid.  The  muscles  were  all  relaxed  and  the  reflexes 


Repair  Processes  of  Protein  Metabolism 


75 


were  all  present.  When  helped  to  his  feet  he  squealed  con- 
stantly and  shifted  rapidly  from  one  hind  foot  to  the  other, 
turning  around  slowly  all  the  time.  When  assisted  to  get 
off  his  feet,  which  he  seemed  unable  to  do  himself,  he  assumed 
the  posture  described  above,  became  quiet  and  was  apparently 
much  easier.  He  appeared  no  worse  during  the  day,  but  at 
five  o ’clock,  when  urged  to  enter  the  cage  he  fell  again  squealing 
loudly,  and  died  an  hour  later. 

Autopsy  revealed  a marked  congestion  of  the  entire  digestive 
tract,  but  much  more  pronounced  in  the  posterior  half.  The  colon 
was  packed  with  putty  like  feces.  Conical  necrotic  areas  were 
found  on  both  kidneys.  Dr.  Hadley,  Station  Veterinarian,  sug- 
gested acute  poisoning,  but  an  examination  of  the  salt  mixture 
failed  to  reveal  the  presence  of  any  toxic  metal  as  was  sus- 
pected. The  casein  used  in  this  experiment  was  made  by  the 
method  of  Hammarsten,  from  separator  skim  milk  from  the 
dairy  building  of  the  Experiment  Station,  and  all  vessels  used 
were  glass. 

The  experiment  was  repeated  on  another  pig  using  ash  of 
whey  instead  of  the  salt  mixture  made  up  from  reagent  bottles. 
The  same  calcium  phosphate  was  given  as  was  used  in  the  first 
experiment,  to  give  increased  body  to  the  feces.  The  second  ex- 
periment was  made  with  a pig  which  had  been  thirty-six  days 
on  a nitrogen-free  diet.  On  the  twenty-fourth  day  of  casein 
feeding,  the  pig  fell  into  the  same  condition  as  the  first,  but  was 
saved  by  promptly  feeding  whole  milk  and  .rolled  oats.  Food 
had  to  be  introduced  into  the  mouth  at  first,  but  after  three  days 
he  was.  on  his  feet  again  and  after  two  weeks  on  the  regular  farm 
ration,  in  an  open  lot,  he  had  gained  12.1  pounds.  When  the 
pig  was  fully  recovered  he  was  again  placed  on  a starch  ration 
and  used  in  a long  continued  experiment  in  low  protein  feed- 
ing. 

It  is  scarcely  warrantable  to  attribute  the  acute  attacks  of 
congestion  which  came  upon  these  pigs,  to  the  casein  fed.  Os- 
borne and  Mendel  report  having  kept  rats  on  a similar  ration 
for  a period  of  160  days  without  serious  disturbances.  The  diffi- 
culty with  these  pigs  is  probably  to  be  attributed  to  the  peculiar 
conformation  of  the  colon  in  the  pig,  and  to  impaction  with 
feces  of  unfavorable  character.  This  point  is  being  further 
investigated. 


76 


Wisconsin  Research  Bulletin  No.  21 


^ Table  VI  shows  a positive  nitrogen  balance  of  206.8  grams. 
I lie  stomach  contained  some  liquid  and  the  intestine  a con- 
siderable quantity  of  fecal  matter,  and  the  tissues  of  the  ani- 
mal  some  end  products  and  partly  metabolized  nitrogenous 
substances.  Experience  with  other  animals  leads  me  to  believe 
that  this  pig’s  body  contained  about  25  grams  of  nitrogen  which, 
if  he  had  been  placed  on  a starch  diet,  would  have  appeared 
in  the  urine  before  the  minimum  level  was  reached.  It  would 
seem  therefore  that  the  pig  had  utilized  for  the  construction 
of  new  body  tissue  about  175  grams  of  nitrogen  taken  as 
casein.  With  this  retention,  of  nitrogen  there  was  a gradual 
rise  in  the  output  of  creatinin  from  .242  grams  per  day  during 
the  first  five  days,  to  .315  grams  during  the  last  five  days. 

The  pig  whose  record  is  shown  in  Table  VII  had,  when  taken 
ill  at  the  end  of  the  experiment,  eaten  259.74  grams  of  nitrogen, 
and  had  excreted  during  the  same  time  152.49  grams.  Since 
this  pig  was  excreting  only  about  4 to  5 grams  of  nitrogen 
daily  in  the  urine  it  is  probable  that  he  would  have  returned 
to  the  minimum  level  of  nitrogen  excretion,  had  he  been  placed 
on  a starch  diet,  with  a loss  of  about  fifteen  grams  of  nitro- 
gen. This  pig  had  retained  and  converted  into  body  tissue 
about  80  to  85  grams  of  casein  nitrogen.  It  is  unfortunate 
that  the  creatinin  record  was  disturbed  by  the  failure  of  the 
pig  to  eat  or  void  any  urine  May  6.  This  makes  it  impossible 
to  arrive  at  a very  satisfactory  figure  for  creatinin  at  the  end 
of  the  experiment.  The  average  for  the  beginning  was  .189 
grams  and  for  the  last  six  days,  after  feeding  casein  .228  grams 
per  day. 

If  we  can  judge  from  the  change  in  the  creatinin  elimination, 
as  to  the  body  content  of  protoplasm,  we  must  conclude  that 
these  pigs  increased  their  metabolizing  tissue  by  an  amount 
equivalent  to  one-fifth  to  one-fourth  on  a diet  containing  but 
a single  protein.  This  is  without  doubt  the  best  evidence  yet 
produced,  of  the  chemical  sufficiency  of  casein  for  both  main- 
tenance and  growth.  These  experiments  with  casein  greatly 
increase  the  value  of  the  negative  results  obtained  in  the  at- 
tempts to  induce  growth  by  feeding  zein  as  the  only  protein. 

Discussion  of  Results 

As  before  stated,  the  experiments  reported  in  this  paper 
were  planned  to  give  an  answer  to  the  question : Why  is  the 


Repair  Processes  of  Protein  Metabolism 


77 


procedure  of  reducing  an  animal  to  its  lowest  possible  level  of 
protein  metabolism  by  feeding  a starch  diet,  then  feeding  an 
amount  of  nitrogen  in  the  form  to  be  studied  equivalent  to  the 
endogenous  metabolism  and  observing  the  excessive  elimina- 
tion of  nitrogen  in  the  urine,  not  a method  for  comparing  the 
relative  values  of  proteins  as  nutrients  for  that  species?  All 
of  my  observations  have  led  to  the  belief  that  zein  or  gelatin 
when  supplying  the  only  source  of  nitrogen  are  useful  in  an 
important  degree,  to  the  animal  in  replacing  the  nitrogen  lost 
through  endogenous  metabolism.  These  “incomplete”  pro- 
teins when  fed  in  liberal  amounts  do  not  seem  to  supply  all  the 
complexes  necessary  for  growth.  The  results  of  Michaud  with 
gliadin  are  substantially  in  harmony  with  mine  for  zein.  In 
his  gliadin  period  Michaud  fed  1.42  grams  of  nitrogen  daily. 
The  average  negative  balance  during  9 days  was  .350  grams 
nitrogen,  equivalent  to  24.64  per  cent  of  the  nitrogen  fed.  In 
his  other  gliadin  periods  the  negative  balances  were  between 
15.05  and  36.33  per  cent  of  the  nitrogen  fed.  Murlin10  found 
gelatin  to  be  utilized  by  dogs  to  the  extent  of  31  per  cent  of 
the  nitrogen  given.  My  results  differ  from  his  only  in  their 
greater  uniformity  due  to  the  fact  that  my  animals  were  re- 
duced to  their  endogenous  level  of  metabolism  in  the  fore 
period  whereas  it  seems  very  doubtful  whether  any  of  his 
were  in  this  condition.  Even  with  a liberal  daily  elimina- 
tion of  urine  I have  not  observed  pigs  to  reach  this  level  on 
a nitrogen-free  diet  in  less  than  sixteen  days. 

The  fact  that  certain  proteins,  lacking  in  one  or  more  cleav- 
age products  known  to  be  necessary  to  the  formation  of  the 
proteins  of  the  animal  body  are  of  relatively  high  efficiency  in 
preventing  loss  of  body  nitrogen  due  to  endogenous  metabolism, 
yet  are  insufficient  for  growth,  forces  one  to  the  conclusion 
that  the  processes  of  replacing  nitrogen  degraded  in  cellular 
metabolism  are  not  of  the  same  character  as  the  processes  of 
growth.  It  seems  also  to  be  a necessary  conclusion  that  the 
processes  of  cellular  catabolism  and  repair  do  not  represent 
a series  of  chemical  changes  involving  the  destruction  and  re- 
construction of  an  entire  protein  molecule.  This  idea  does  not 


IQ  Murlin,  Amer.  Jour,  of  Physiol.  20,  240  (1907), 


78 


Wisconsin  Research  Bulletin  No.  21 


conflict  with  the  theory  of  protein  metabolism  offered  by  Folin.11 
Osborne  and  Mendel12  have  recently  shown  that  certain  pro- 
teins may  support  an  animal  in  nitrogen  equilibrium  for  long 
periods  and  yet  be  unable  to  produce  growth.  My  experience 
in  feeding  casein  as  the  only  protein  has  convinced  me  that 
a marked  increase  in  the  protein  content  of  the  body  may  re- 
sult from  taking  a single  protein  in  the  food.  The  facts  there- 
fore lead  to  the  belief  that  in  order  that  an  animal  may  grow, 
the  food  protein  must  supply  complexes  not  necessary  for  the 
endogenous  upkeep. 

It  is  interesting  to  correlate  the  results  of  these  experiments 
with  the  findings  of  Folin* 1*  in  his  studies  of  the  fate  of  creatin 
when  fed  to  men  on  a low  protein  diet.  He  was  unable  to 
trace  the  nitrogen  of  this  complex  into  the  urine  under  these 
conditions  and  was  led  to  conclude  that  creatin  acts  as  a food 
and  not  as  an  end  product  of  metabolism. 

These  fragments  of  evidence  do  not  harmonize  with  the  views 
of  Michaud,14  which  have  been  endorsed  by  Aberhalden.15 
In  fact  it  seems  rather  surprising  that  Michaud  should  draw 
the  conclusion  quoted  early  in  this  paper  when  he  could  trace 
but  15  to  36  per  cent  of  the  nitrogen  of  gliadin  into  the  urine 
m his  feeding  experiments  with  this  protein  known  to  be 
lacking  m one  cleavage  product,  lysin,  present  in  all  animal 
proteins  examined.  According  to  his  theory  this  protein 
should  be  without  value  to  the  animal,  so  far  as  its  nitrogen 
is  concerned,  if  fed  alone. 

Summary  of  Conclusions 

Attention  is  called  to  the  fact  that  the  results  of  experi- 
ments in  feeding  the  mixture  of  proteins  occurring  in  individ- 
ual grams,  in  quantity  equivalent  to  the  lowest  possible  level  of 
protein  metabolism  of  which  the  animal  is  capable,  do  not  in- 
dicate as  wide  differences  in  the  nutritive  values  of  the  pro- 
tein of  the  wheat,  oat,  and  corn  kernels  as  would  be  expected 
from  the  known  chemical  differences  in  these  proteins. 


ii  Amer.  Jour.  Physiol.  1(5,  117  (1905). 
is  Carnegie  Inst.  Bui.  156  (1911). 
i s Festschrift  fur  Olaf  Hammarsten,  1906. 

14  Michaud,  Ztschr.  Physiol.  Chem.  39,  405  (1909). 

15  Ztschr.  Physiol.  Chem.  60,  425  (1909). 


Repair  Processes  of  Protein  Metabolism 


79 


Experiments  are  described  in  feeding  zein  and  gelatin,  two 
proteins  which  are  ‘ 'incomplete”  chemically,  in  that  they 
lack  certain  cleavage  products  known  to  be  present  in  animal 
proteins.  It  is  shown  that  the  animal  can  utilize  the  nitrogen 
of  zein  very  efficiently  for  repair  of  the  losses  due  to  endog- 
enous or  tissue  metabolism.  The  average  utilization  of  zein 
nitrogen  for  this  purpose,  was  about  80  per  cent,  for  gelatin 
50  to  60  per  cent.  Nq  evidence  was  obtained  of  the  formation 
of  additional  body  tissue  from  zein,  even  when  the  latter  was 
fed  in  great  excess  over  the  maintenance  needs  of  the  animal. 

Experiments  in  feeding  casein  as  the  only  protein,  resulted 
in  increases  of  the  body  protein  of  20  to  25  per  cent.  These 
are  the  most  successful  growing  experiments  yet  reported  in 
which  but  a single  protein  was  fed. 

The  experimental  data  presented  do  not  harmonize  with  the 
most  widely  accepted  theories  concerning  the  mechanism  of 
protein  metabolism.  The  repair  processes  are  shown  to  be  of 
a different  character  from  the  processes  of  growth.  The  re- 
sults of  the  work  here  presented  are  believed  to  indicate  that 
the  processes  of  cellular  catabolism  and  repair  do  not  involve 
the  destruction  and  resynthesis  of  an  entire  protein  molecule. 


80 


Wisconsin  Research  Bulletin  No.  21 


A METABOLISM  CAGE  FOR  THE  PIG 

E.  V.  McCOLLUM  and  H.  STEEN  BOCK 

During  the  past  four  years  in  this  laboratory,  pigs  have  been 
in  use  continuously  as  experimental  animals  in  nutrition 
studies.  They  have  proven  especially  satisfactory  in  certain 
respects.  Almost  all  of  our  exact  knowledge  of  the  chemis- 
try of  nutrition  has  been  gained  through  studies  conducted 
with  carnivora  and  herbivora,  in  each  of  which  types  dietary 
habits  and  a number  of  well  marked  differences  in  metabolic 
processes  depart  widely  from  the  human. 

In  the  pig  the  plan  of  the  nutritional  processes  is  closely 
similar  to  that  in  man.  In  marked  contrast  to  man,  the  per- 
iod of  infancy  and  therefore  of  growth  are  exceedingly  short 
for  the  great  size  attained.  The  pig  is  probably  endowed 
with  a greater  ability  to  utilize  food  for  growth  than  any 
other  animal  large  enough  for  satisfactory  quantitative  col- 
lections of  the  urine  during  early  life  when  growth  is  most 
rapid.  Added  to  this  are  its  indolent  habits  which  en- 
able it  to  remain  closely  confined  for  long  periods  without 
chafing,  and  its  almost  unfailing  appetite  even  for  food  of 
low  palatability.  It  almost  never  regurgitates  its  food,  and  if 
freed  from  intestinal  parasites  is  not  subject  to  digestive  dis- 
turbances. 

One  of  us  has  noted  elsewhere  that  a pig  will  take  for  long 
periods  a liberal  energy  supply  in  the  form  of  pure  starch, 
scalded  and  made  into  a thin  soup,  with  salt  mixture  supply- 
ing all  necessary  mineral  elements.  (See  page  76).  This  ani- 
mal offers,  therefore,  special  advantages  for  the  study  of  many 
problems  connected  with  protein  metabolism. 

In  the  progress  of  our  work  a metabolism  cage  has  been 
evolved  suitable  for  the  quantitative  collection  and  separ- 
ation of  the  excreta  of  the  pig.  Since  this  seems  now  to  be 
perfected,  we  have  thought  it  advisable  to  describe  its  con- 
struction and  operation. 


# 


A Metabolism  Cage  for  the  Pig 


81 


Construction  of  the  Cage 

The  cage  is  shown  in  all  essential  details  in  the  accompany- 
ing illustrations.  It  consists  of  a main  cage  in  which  the 
animal  is  confined  except  when  being  fed  and  a feeding  stall 
which  communicates  with  the  main  cage  by  the  sliding  doors 
N and  C.  The  large  retaining  cage  as  shown  to  the  right  in 
Figure  1 consists  of  a square  zinc  lined  box  having  a hinged 


FIGURE  1.  METABOLISM  CAGE  FOR  PIG,  RIGHT  SIDE 


side,  G,  a hinged  iron  barred  top,  a dividing  partition  F,  and 
a screen  floor  D.  The  whole  is  supported  on  four  iron  castors 
which  enable  the  cage  to  be  rolled  along  a wooden  track  J, 
either  over  the  zinc  topped  draining  table  or  over  the  acces- 
sory table  I,  exposed  in  the  picture.  The  walls  of  the  cage 
are  zinc  covered  to  within  4 1-2  inches  of  the  bottom  of  the 
cage  at  which  place  they  are  beveled  inwardly  2 1-2  inches. 
The  hinged  side  G,  is  also  zinc  covered  and  beveled  as  shown 
by  A,  like  the  other  wall.  This  side  serves  as  the  door 
through  which  the  animal  can  be  introduced  and  solid  excreta 
removed.  The  top  of  the  cage  consists  of  a grating  formed 
of  % inch  iron  bars  held  in  a wooden  2x4  inch  frame.  From 
these  bars,  by  means  of  hooks,  the  zinc  covered  dividing  parti- 


82 


Wisconsin  Research  Bulletin  No.  21 


tion  F,  of  the  cage  is  suspended.  This  partition  makes  it  possi- 
ble to  adjust  the  size  of  the  cage  for  different  sized  animals 
and  thus  avoids  the  needless  exposure  of  floor  space. 

The  floor  of  the  cage  consists  of  a heavy  screen  four  meshes 
to  the  inch  of  number  11  wire  which  is  fastened  by  means  of 
staples  to  a 2x4  inch  frame  and  further  supported  by  horizon- 
tal iron  bars  %xl  inch  at  15  inch  intervals.  These  bars  are 
likewise  inserted  in  the  2x4  inch  frame  which  is  introduced 
from  the  bottom  of  the  cage  up'  to  within  % inch  of  the  bev- 
eled edges  of  the  zinc  covered  walls.  The  urine  as  voided 
passes  through  the  screen  to  the  draining  pan  B in  Figure  2, 


FIGURE  2.  CROSS  SECTION  OF  METABOLISM  CAGE  FOR  PIG 


which  is  zinc  covered  and  drains  to  its  center  with  a fall  of 
two  inches  to  the  foot  dropping  into  receptacle  C.  The  space 
of  % inch  between  the  beveled  lower  edges  of  the  zinc  cover- 
ed wall,  and  the  screen  floor  serves  a double  purpose.  In  the  first 
place  it  obviates  the  presence  of  angles  into  which  feces  might  be- 
come lodged  and  from  which  they  could  be  removed  with  diffi- 
culty. With  this  construction  all  feces  can  be  readily  removed 
from  the  screen  floor.  The  periphery  of  the  screen  floor  is  never 
in  contact  with  feces,  and  this  portion  is  easily  gotten  at  for 
brushing  and  cleaning  by  turning  the  cage  on  its  side  so  that 
the  bottom  is  accessible.  In  the  second  place  it  permits  the 
superimposition  of  a second  lighter  screen  over  the  screen 
floor  and  below  the  beveled  edges  of  the  walls.  The  screen 
is  shown  at  D in  Figure  1.  Between  these  screens  is  placed 
a single  thickness  of  cheese  cloth  stretched  over  the  upper 


A Metabolism  Cage  for  the  Pig 


83 


screen  by  perforating  the  corners  of  the  cloth  by  the  corners 
of  the  screen. 

This  last  device  has  been  employed  only  when  rations  rich 
in  wheat  bran  were  used.  Such  rations  produce  bulky  and 
granular  feces  which  readily  fall  through  the  screen  floor 
upon  the  draining  pan,  the  slope  of  which  facilitates  distri- 
bution and  contamination  with  urine.  In  all  ordinary  work 
this  device  has  not  been  used  except  as  an  easy  means  of  keep- 
ing the  cage  clean  when  no  collections  were  being  made  while 
a pig  was  becoming  familiar  with  the  cage. 


Facilities  for  Feeding  the  Pig 

The  auxiliary  feeding  cage  is  best  shown  in  Figures  3 and 
4.  It  consists  essentially  of  a narrow  box  fitted  by  means 


FIGURE  3.  METABOLISM  CAGE  FOR  PIG,  LEFT  SIDE 


of  cleats  on  the  inside  to  the  supporting  bench.  The  latter  is 
fitted  in  the  rear  with  a sliding  zinc  lined  drawer  which  passes 
under  a heavy  screen  D,  which  makes  up  the  rear  part  of  the 
floor  in  the  cage.  This  screen  is  of  the  same  construction  as 
the  one  previously  described  in  connection  with  the  main 
cage.  This  screen  and  the  sliding  drawer  beneath  serve  as  a 


84 


Wisconsin  Research  Bulletin  No.  21 


safety  device  in  case  the  pig  should  void  any  urine  when  in  the 
feeding  stall.  In  practice  this  almost  never  happens  if  the  pig 
is  left  in  the  stall  only  while  busy  eating. 

The  feeding  cage  proper  consists  of  a narrow  box  fitted  with 
a sloping  floor  and  beveled  tin  lined  walls  so  as  to  drain  into 
the  drawer  below.  The  rear  trap  door,  C,  communicates  with 
the  lar?e  cage  while  through  the  front  hinged  door  M,  the  ani- 
mal can  be  fed  in  the  galvanized-iron-lined  trough  E.  As 
required,  the  trough  may  be  slid  sideways  and  removed  from 


the  cage  for  cleaning.  To  prevent  the  animal  from  scatter- 
ing his  feed  by  turning  around  while  feeding,  the  cage  is  pro- 
vided with  a movable  partition  P,  which  fits  in  between 
cleats  and  thus  can  be  adjusted  to  confine  an  animal  as  closely 
as  desired.  The  part  of  the  floor  on  which  the  pig’s  fore  feet 
stand,  H,  in  Figure  5 is  zinc  lined  to  facilitate  cleaning. 

The  special  difficulties  connected  with  the  feeding  of  a pig 
Kept  in  a metabolism  cage  are  obviated  by  transferring  the 
animal  to  the  feeding  stall  at  meal  time.  Advantage  should 
be  taken  of  the  absence  of  the  pig  from  the  main  cage,  for 
cleaning  the  latter  at  this  time.  The  sliding  doors  N and  C 


A Metabolism  Cage  for  the  Pig 


85 


are  lifted  and  the  pig  will  of  his  own  accord  after  a brief 
period,  hasten  to  the  trough  E in  which  the  ration  has  been 
placed.  The  doors  are  allowed  to  drop  and  at  once  the  hinged 
side  G is  lowered  to  give-  access  to  the  cage.  The  feces  are  re- 
moved so  far  as  possible  by  means  of  a broad  steel  spatula  and 
the  remainder  are  brushed  through  upon  the  draining  pan  by 
means  of  a strong  steel  brush.  The  cage  is  then  pushed  along 
the  track  J,  so  that  the  draining  pan  can  be  carefully  brushed 
free  from  feces,  the  latter  being  collected  in  a pan.  Previous  to 
washing  the  screen  floor  of  the  cage  it  is  our  custom  to  tip  the 
cage  upon  its  side  and  thoroughly  brush  the  bottom  with  the 
steel  brush.  When  the  pan  is  free  from  feces  the  cage  and 
pan  are  washed  with  the  usual  precautions  in  such  work. 


This  can  usually  be  done  in  the  time  during  which  the  pig  will 
busy  himself  with  cleaning  up  the  trough.  He  is  transferred 
again  to  the  cage  when  all  is  ready. 

We  have  found  it  of  advantage,  to  use  only  male  pigs  for 
cage  work  since  with  these  the  urine  is  deposited  within  a 
narrow  radius  near  the  center  of  the  floor  and,  the  cage  being 
adjusted  to  just  permit  the  animal  to  turn  around  and  to 
lie  stretched  out  when  placed  diagonally,  the  feces  will  al- 
ways tend  to  be  deposited  near  the  walls. 

Another  important  matter  is  that  of  clipping  the  hair  from 
the  head  of  the  animals.  This  prevents  food  from  adhering 
to  the  face.  It  is  a simple  matter  to  wipe  any  adhering 
particles  from  the  nose  and  face  with  a small  cloth  before 
replacing  in  the  main  cage  after  eating. 

In  work  with  pigs  confined  in  the  cage  we  have  found 
it  advantageous  to  modify  the  character  of  the  feces  by  giv- 
ing either  agar-agar  or  calcium  phosphate  or  both.  By  this 


86 


Wisconsin  Research  Bulletin  No.  21 


means  the  successful  operation  of  the  cage  without  contam- 
inating the  urine  is  rendered  much  more  easy  and  certain. 

The  successful  operation  of  the  cage  requires  visiting  it 
several  times  a day  ,so  as  not  to  allow  the  accumulation  of 
feces  on  the  floor  for  any  great  length  of  time.  With  care, 
however  it  gives  good  results. 


Metabolic  Water:  Its  Production  and  Role 


in  Vital  Phenomena 


S.  M.  BABCOCK 

Water  is  essential  to  life  and  during  the  period  of  develop- 
ment it  is  the.  most  abundant  constituent  of  living  organisms, 
its  amount  ranging  from  about  40  to  nearly  100  per  cent  of  the 
total  weight.  Some  of  this  water  is  imbibed  directly,  some  of 
it  is  taken  with  the  solid  food  which  is  ra,rely  dry,  and  some  of 
it  is  formed  within  the  organism  by  metabolic  changes  in  the 
organic  constituents  of  the  food  and  tissues,  induced  by  respira- 
tion and  other  vital  processes.  The  relative  amount  of  water 
derived  from  each  of  these  sources  depends  upon  the  kind  of 
organism,  its  period  of  growth,  the  nature  of  its  food,  its  envi- 
ronment, and  its  activities. 

The  chief  functions  of  water  are  to  dissolve  nutrients  and 
serve  as  a medium  for  their  distribution,  to  remove  injurious 
waste  products  from  the  cells,  to  control  the  temperature,  within 
narrow  limits,  by  its  evaporation,  and  in  chlorophyl  producing 
plants  to  supply  material  for  the  synthesis  of  organic  matter. 

"~Tn  general,  plants  and  most  animals  require  an  abundant 
and  frequent  supply  of  water  from  external  sources  at  all  peri- 
ods of  growth  in  order  that  these  functions  may  be  properly 
performed.  * There  are,  however,  particular  stages  in  the  life 
history  of  both  plants  and  animals  in  which  metabolic  water 
is  sufficient  for  all  purposes,  for  considerable  periods  of  time. 
Thus  in  the  resting  periods  of  deciduous  plants,  in  bulbs,  in 
tubers,  and  especially  in  seeds  and  spores  ample  water  for  all 
vital  processes  is  provided  by  the  slow  oxidation  that  takes  place 
as  a result  of  direct  respiration.  This  is  also  true  in  the  case 
of  hibernating  animals  that  receive  no  water  from  external 


88 


Wisconsin  Research  Bulletin  No.  22 


sources  for  several  months,  although  water  is  constantly  lost 
through  respiration  and  the  various  excretions.  In  addition, 
many  varieties  of  Insects  such  as  the  clothes  moths,  the  grain 
weevils,  the  dry  wood  borers,  etc,  are  capable  of  subsisting,  dur- 
ing all  stages  of  development,  upon  air  dried  food  materials  con- 
taining less  than  ten  per  cent  of  water;  in  these  cases,  nearly  all 
of  the  water  required  is  metabolic. 

^ While  the  production  of  metabolic  water  has  been  recognized 
by  all.  students  of  plant  and  animal  physiology,  there  has  here- 
tofore been  no  distinction  made  between  its  functions  and  those 
of  imbibed  water.  It  has  evidently  been  assumed,  because  the 
molecular  structure  of  water  from  various  sources  is  the  same, 
that  it  always  serves  the  same  purposes  in  vital  processes, 
whether  derived  from  external  or  internal  sources. 

It  is  the  purpose  of  this,  paper  to  show  that  metabolic  water 
is  not  only  produced  in  considerable  quantity  from  the  organic 
constituents  of  the  food  and  tissues  of  plants  and  animals  by 
oxidation  and  by  dehydrating  reactions,  but  also  that  water  so 
produced  performs  a different  function  from  imbibed  water  and 
m many  instances,  if  not  in  all,  is  essential  to  the  growth  and 
continued  life  of  the  organism. 

^ ' Sources  of  Metabolic  Water 

The  most  obvious  source  of  metabolic  water  is  the  oxidation  of 
organic  matter  comprising  the  food  and  tissues  of  an  organism, 
by  means  of  free  oxygen  derived  from  the  air  during  respira- 
tion. This  production  of  water  is  always  associated  with  an 
absorption  of  free  oxygen  and  an  evolution  of  carbon  dioxide, 
the  carbon  dioxide  being  of  practically  the  same  volume  as  the 
jibsorbed  oxygen. 

Many  organisms  also,  when  deprived  of  free  oxygen,  are  cap- 
able of  maintaining,  for  a short  time,  certain  of  the  respiratory 
functions,  and  deriving  energy  from  food  material  and  from 
tissues  by  breaking  up  the  molecular  structure  into  new  forms 
of  a lower  order.  This  is  known  as  intramolecular  respiration 
and  like  direct  respiration,  results  in  the  production  of  both 
water  and  carbon  dioxide. 

Metabolic  water  is  also  produced  by  all  organisms  through 
changes  in  the  molecular  structure  of  substances  composing  its 
nutrients  or  its  tissues.  The  transformation  of  dextrose  or  in- 


Role  of  Metabolic  Water  in  Vital  Phenomena  89 


vert  sugar  into  cellulose,  starch,  or  cane  sugar,  and  the;  forma- 
tion of  muscular  fiber,  and  other  complex  proteids  from  pep- 
tones or  from  amino  acids,  are  examples  of  such  reactions.  No 
carbon  dioxide  is  evolved  in  changes  of  this  nature.  This  source 
of  metabolic  water  is  probably  the  most  important  of  all,  if  ag- 
gregate quantities  are  alone  considered,  since  the  same  carbon 
nucleus  may  function  alternately  in  hydrolytic  and  dehydrating 
reactions  an  indefinite  number  of  times. 

Respiration , either  direct  or  intramolecular  with  its  consequent 
loss  of  organic  matter  and  evolution  of  carbon  dioxide  continu- 
ally takes  place  in  living  organisms  of  all  kinds  and  its  total 
suspension,  even  for  a brief  period,  is  the  best  evidence  of  death. 
This  becomes  more  apparent  when  it  is  realized  that  the  energy 
required  for  maintaining  vital  activity  of  all  kinds  is,  in  its 
final  analysis,  wholly  derived  from  the  slow  combustion  of  or- 
ganic substances,  stored  within  the  organism. 

The  phenomena  of  photosynthesis,  which  enables  green  plants 
to  utilize  solar  energy  for  the  accumulation  of  food  materials 
from  which  tissue  is  formed,  a, re  not  opposed  to  this  view  since 
the  further  use  of  such  substances  by  a plant  requires  a consider- 
able expenditure  of  energy  which  can  be  derived  only  from  oxi- 
dation or  other  degradation  of  substances  already  formed.  The 
function  of  these  stored  materials  in  promoting  the  vital  activity 
of  a plant  is  entirely  analogous  to  that  of  food  materials  sup- 
plied to  the  digestive  tract  of  an  animal,  which  serve  no  useful 
purpose  for  the  animal  until  they  are  digested  and  assimilated. 
Photosynthesis  is  a means  of  collecting  food  material  which, 
when  assimilated  and  incorporated  into  the  cellular  structure 
of  a plant,  may  be  oxidized  and  a portion  of  its  stored  energy 
utilized  for  maintaining  the  vital  processes.  Until  these  ma- 
terials become  a part  of  the  cellular  structure,  or  of  the  cir- 
culatory fluids,  they  can  contribute  no  energy  to  the  support 
of  an  organism.  Solar  energy  converts  compounds,  which  the 
plant  is  not  able  to  use  directly,  into  raw  food  materials.  Assim- 
ilation of  this  raw  material  is  only  accomplished  by  destruc- 
tion of  material  previously  assimilated.  These  changes  are  all. 
directly  or  indirectly,  dependent  upon  energy  derived  from  com- 
bination of  organic  nutrients  with  oxygen  acquired  by  respira- 
tion. 

Water  Derived  from  Oxidation  of  Nutrients  and  Tissue  The 
substances  oxidized  by  both  plants  and  animals,  to  supply  vital 


90 


Wisconsin  Research  Bulletin  No.  22 


energy,  consist  chiefly  of  carbohydrates,  fats,  and  proteins.  All 
of  these  substances  contain  hydrogen,  and  their  complete 
oxidation  produces  a quantity  of  water  equal  to  nine  times  the 
weight  of  hydrogen  present  in  the  original  substance.  Thus  one 
hundred  parts  of  cellulose  or  starch,  (C6H10OB)n,  containing 
6.17  per  cent  of  hydrogen,  gives  55.5  parts  of  water;  one  hun- 
dred parts  of  anhydrous  dextrose  containing  6.66  per  cent  of 
hydrogen  gives  60  parts  of  water  etc.  Most  of  the  fats  yield 
more  than  their  weight  of  water,  while  proteins,  when  com- 
pletely oxidized,  give  from  60  to  65  per  cent  of  water.  Protein 
metabolism,  however,  is  not  a factor  that  affects  the  total  water 
content  of  a plant,  since  in  general,  the  nitrogenous  substances 
resulting  from  respiration  are  again  assimilated,  as  much  water 
being  absorbed  in  their  reconstruction  as  was  set  free  in  their 
breaking  down.  On  the  other  hand,  protein  metabolism  may  be 
an  important  factor  in  the  transfer  and  distribution  of  water 
from  place  to  place,  since  the  destructive  and  synthetic  reactions 
often  occur  in  cells  quite  remote  from  each  other.  Animals, 
however,  are  unable  to  utilize  the  final  products  of  protein  meta- 
bolism which  are  in  most  cases  poisonous  and  must  be  removed 
from  the  tissues  by  excretion  in  various  forms,  the  principal  of 
which  are  urea,  uric  acid,  and  ammonia.  The  amount  of  meta- 
bolic water  derived  from  the  breaking  down  of  protein,  when 
urea  in  excreted,  is  about  42  per  cent  of  the  weight  of  protein, 
when  uric  acid  is  excreted  nearly  53  per  cent,  and  when  am- 
monia is  excreted  about  32  per  cent. 

The  amount  of  metabolic  water  formed  by  oxidation  during 
any  period  is  proportional  to  the  rate  of  respiration.  Every 
circumstance  which  hastens  or  .retards  respiration  also  affects,  in 
the  same  way,  the  production  of  metabolic  water.  The  rate  of 
respiration  differs  with  the  type  of  organism,  even  though  ex- 
ternal conditions  may  be  the  same.  As  a rule  it  is  slower  with 
plants  than  with  animals,  and  slower  with  cold  blooded  than  with 
warm  blooded  animals.  It  varies  widely  for  the  same  indivi- 
dual at  different  stages  of  development  and  even  in  different 
organs  of  the  same  organism  at  the  same  time.  In 'dry  seeds 
and  spores  respiration  is  practically  suspended,  it  being  possible 
to  detect  it  only  by  observations  extending  over  long  periods  of 
time ; it  is  also  slow  in  bulbs  and  tubers,  and  in  the  whole  plant 
during  the  winter;  it  is  most  pronounced  when  vital  processes 


Hole  op  Metabolic  Water  in  Vital  Phenomena  91 

are  most  active,  as  during  the  germination  of  seeds,  at  flowering, 
and  at  other  periods  of  rapid  growth. 

Intracellular  Production  of  Metabolic  Water  Respiration  is 
a function  peculiar  to  active  protoplasm.  Since  this  substance 
which  constitutes  the  physical  basis  of  all  life  is,  with  minor  ex- 
ceptions, organized  as  distinct  cellular  units,  it  follows  that  the 
production  of  metabolic  water  is  mostly  confined  to  the  interior 
of  such  cells. 

In  consequence  of  this  local  production  of  water,  the  concen- 
tration of  the  cell  contents  is  reduced,  both  by  the  production 
of  water  and  by  the  elimination  of  soluble  organic  matter.  This 
change  in  concentration  disturbs  the  osmotic  equilibrium  be- 
tween the  fluids  within  and  without  the  cell  and  a movement  of 
nutrients  by  osmosis  is  induced  towards  the  depleted  centers.  It 
is  not  an  essential  condition  for  these  effects  that  soluble  nutri- 
ents within  a cell  be  completely  oxidized  I it  is  sufficient  if  the 
molecular  structure  of  nutrients  within  a cell  be  changed,  es- 
pecially if  the  change  results  in  the  liberation  of  water,  or  if 
the  nutrients  be  rendered  insoluble,  as  occurs  when  soluble  car- 
bohydrates are  deposited  in  the  form  of  starch,  cellulose,  or  fat, 
or  when  soluble  and  diffusible  nitrogen  compounds  are  converted 
into  insoluble  tissue  or  even  into  non-diffusible  proteins. 

Water  from  an  external  source  does  not  serve  these  purposes, 
since  its  immediate  effect  is  to  reduce  the  concentration  of  solu- 
ble nutrients  in  the  circulatory  fluids  to  a point  below  that  in  the 
fluids  of  the  cells  and  thus  cause  a movement  of  nutrients  away 
from,  rather  than  towards  the  points  where  they  are  most  need- 
ed. If  the  distribution  of  nutrients  is  alone  considered,  ab- 
sorbed water  tends  towards  the  starvation  of  the  cells.  Absorbed 
water  does,  however,  facilitate  the  removal  of  waste  products, 
replaces  water  lost  through  evaporation  and,  in  the  case  of  chlo- 
rophyl  producing  plants,  supplies  material  for  the  synthesis  of 
organic  nutrients  and,  for  this  reason,  an  abundant  supply  of 
water  from  external  sources  is  absolutely  essential  for  the  growth 
of  this  class  of  plants.  With  parasitic  plants,  and  with  animals, 
which  derive  all  of  their  organic  nutrients  from  chlorophyl  pro- 
ducing plants,  imbibed  water  is  not  so  essential  to  life;  with 
these  the  chief  function  of  imbibed  water  is  to  aid  in  the  re- 
moval of  waste  products,  the  metabolic  water  being  in  most 
cases  sufficient  for  transferring  nutrients  and  for  replacing  the 
ordinary  losses  incurred  by  respiration  and  evaporation. 


92 


Wisconsin  Research  Bulletin  No.  22 


Metabolic  Water  in  Seeds1 

A seed  consists  of  an  embryo  plant  in  a nearly  dormant  con- 
dition, surrounded  by  sufficient  reserve  food  material  to  nourish 
it  until  it  has  reached  a stage  of  development  in  which  it  is  cap- 
able of  assimilating  carbon  dioxide  from  the  air,  and  inorganic 
substances  from  the  soil. 

Although  the  embryo  of  an  air  dried  seed  is  generally  assumed 
to  be  entirely  dormant,  it  continues  to  perform  certain  vital  func- 
tions similar,  except  in  degree,  to  those  of  a mature  plant.  Chief 
among  these  is  respiration  which  is  manifested  by  an  absorption 
of  oxygen,  an  evolution  of  carbon  dioxide,  the  production  of 
water,  and  a consequent  loss  of  dry  matter. 

Water  Content  of  Seeds  Since  the  production  of  water  from 
the  oxidation  of  organic  matter  is  a necessary  consequence  of  re- 
spiration, all  viable  seeds  must  contain  some  free  water  at  all 
times.  The  water  content  of  air  dried  seeds  of  most  economic 
plants,  ranges  from  about  five  to  more  than  twenty  per  cent, 
depending  upon  the  degree  of  saturation  of  the  surrounding  air! 
For  most  of  the  common  grains  it  is  about  ten  per  cent. 

It  is  impracticable  to  remove  all  water  from  seeds  except  by 
prolonged  exposure  to  a temperature  approximating  100°  C. 
Seeds  dried  in  this  way  and  again  exposed  to  ordinary  air,  even 
though  it  be  moderately  dry,  absorb  from  five  to  ten  per  cent  of 
moisture  in  a short  time.  Seeds  kept  in  a saturated  atmosphere 
for  three  or  four  weeks  usually  contain  over  30  per  cent  of 
water  and  if  temperature  conditions  are  favorable  such  seeds 
will  germinate  without  having  been  in  direct  contact  with  water 
or  with  a moist  surface.  Between  these  limits  seeds  may  con- 
tain any  proportion  of  water  depending  upon  the  degree  of 
saturation  of  the  air  and  the  time  of  exposure.  The  strong  af- 
finity of  seeds  for  water  and  the  persistence  with  which  the  ab- 
sorbed water  is  retained  at  ordinary  temperatures  indicate  that 
there  is  a feeble  molecular  combination  of  water  with  substances 
comprising  the  seed  analogous  to  that  occurring  in  crystals  con- 
taining water  of  crystallization.  There  is  also  little  doubt  that 
the  physical  structure  of  a seed,  especially  of  its  outer  covering 

i Unless  otherwise  stated,  the  observations  upon  seeds  recorded  in 
this  paper,  refer  to  corn,  (Zea  mays);  it  is  believed,  however  that 
they  apply  to  other  varieties  of  seeds  as  well.  Corn  has  been  se- 
lected for  the  tests  because  of  the  large  seed  and  the  ease  with 
which  the  embryo  may  be  separated  from  other  parts  of  the  germi- 
nating kernel. 


Role  of  Metabolic  Water  in  Vital  Phenomena 


93 


or  hull,  is  an  important  facto.r  affecting  both  the  absorption  and 
retention  of  water.  The  influence  which  the  hull  exerts  upon 
the  retention  of  water  is  illustrated  by  Table  I,  in  which  the 
times  required  for  drying  whole  kernels  of  corn  and  kernels  from 
the  same  ear,  with  the  hull  cut  through  in  a number  of  places 
with  a sharp  knife,  and  other  kernels  divided  into  particles  of 
different  size,  are  compared. 

The  same  number  of  kernels  and  approximately  the  same 
weight  of  material  was  dried  in  each  case  in  a steam  oven  the 
temperature  of  which  was  approximately  97°  C.  It  should  be 
borne  in  mind  that  at  this  temperature  drying  proceeds  at  a 
considerably  lower  rate  than  at  100°  C.  Fine  corn  meal  dries 
to  practically  constant  weight  in  10  to  12  hours,  at  this  tem- 
perature. 

TABLE  1.  INFLUENCE  OF  HULL  ON  ESCAPE  OF  MOISTURE 
Kernels  of  corn  (Zea  mays ) were  dried  at  97°  C. 


Percentage  Loss  in  Weight 


Hours  exposed 

Whole 

kernels 

Kernels  with 
broken  hulls 

I 

Split 

kernels  j 

Meal 

5 

4.47 

6 04 

6.20 

7.94 

24  

6.85 

8.04 

7.95 

8.89 

30  

7.07 

8.22 

8.12 

8.97 

48 

7.68 

8.54 

8.52 

72 

8.18 

8.77 

8,68 

96 

8.47 

8.95 

8.90 

120 

8.76 

114 

8*89 

168 

9.01 

It  appears  from  the  table  that  split  kernels  and  those  with 
only  the  hulls  cut  through,  dry  at  practically  the  same  rate,  but 
this  is  considerably  slower  than  with  rather  coarse  meal.  The 
difference  in  the  rates  of  drying  of  these  kernels  and  of  the 
meal,  is  best  explained  by  the  relatively  larger  surface  exposed 
in  the  particles  of  meal.  On  the  other  hand,  the  kernels  with 
unbroken  hulls  have  approximately  the  same  surface  as  the 
others  but  require  a materially  longer  time  to  lose  the  same 
weight,  at  97°  C.  This  difference  must  be  attributed  to  the  pro- 
tecting influence  of  the  hull.  Similar  results  have  been  ob- 
tained in  a great  number  of  tests  in  which  whole  kernels  of  corn 
have  been  dried  at  97°  0.  In  practically  all  such  trials  not  less 


94 


Wisconsin  Research  Bulletin  No.  22 


than  168  Honrs  have  been  necessary  to  .reduce  the  loss  in  weight 
to  less  than  .1  per  cent  in  twenty-four  hours,  whereas  meal  or 
broken  kernels,  containing  as  much  moisture,  have  dried  to  the 
same  extent  in  about  twenty-four  hours. 

The  persistence  with  which  whole  kernels  of  corn  retain  mois- 
ture is  still  better  illustrated  in  an  experiment  designed  to  show 
the  effects  of  respiration  and  the  production  of  metabolic  water 
in  air  dried  corn.  In  this  experiment,  corn  was  placed  in  a des- 
iccator over  sulphuric  acid  and  the  loss  of  weight  determined 
after  varying  intervals.  Samples  from  the  same  lot  were  dried 
in  a steam  oven,  to  practically  constant  weight,  at  the  beginning 
of  the  test,  and  afterwards,  corn  from  the  desiccator,  was  dried 
in  the  same  manner  at  dates,  when  observations  for  weight  were 
made.  The  percentages  given  in  Table  II  are  all  calculated  up- 


TABLE  II.  INFLUENCE  OF  DRY  AIR  ON  LOSS  OF  MOISTURE 

Loss  of  weight  of  corn  exposed  to  a temperature  of  97°C.  compared  with  that  of 
corn  kept  in  a desiccator  over  sulphuric  acid. 


Dates  Weighed 

Days 
of  Test 

Per  Cent  Loss  in  Weight 

Over  HsSO* 

1 

At  97°C. 

Total 

1908. 

May  9 

0 

8.42 

8.42 

Aug.  21 

Oct  tc; 

104 

6.15 

1909. 

159 

6.64 

Feb.  20 

287 

7.32 

1.27 

8.59 

May  4 

360 

7.48 

1.20 

8.68 

Sept.  28 

507 

7.92 

0.97 

8.89 

1910. 

Feb.  21 

653 

8.13 

0.90 

9.03 

May  13 

734 

8.25 

0.84 

9.09 

Sept.  8 

852 

8.36 

0.74 

9.10 

1911. 

Feb.  7 

1004 

8.53 

0.71 

9.24 

May  9 

1095 

8.56 

0.39 

8.95 

Sept.  18 

1227 

8.69 

0.00 

1 

8.69 

on  the  weight  of  the  air  dried  sample  at  the  beginning  of  the 
test.  The  corn  used  was  a white  dent  variety  of  the  crop  of 
1907 ; it  was  well  cured  as  shown  by  a number  of  germination 
tests  including  more  than  100  kernels  in  which  every  kernel 
germinated  with  strong  healthy  sprouts;  it  was  placed  in  the 
desiccator  May  9,  1908. 

If  it  be  assumed  that  the  loss  in  weight  at  97°  C.  represents 
water  only,  and  that  the  organic  matter  of  the  seed  is  practic- 
ally non-volatile,  at  the  .room  temperatures  in  which  the  desicca- 


Role  of  Metabolic  AVater  in  Vital  Phenomena  95 


tor  containing  the  corn  was  kept,  there  is  still  considerable  water 
remaining  in  this  corn  at  the  end  of  3%  years.  In  any  case 
there  has  been  a gradual  and  continual  loss  of  dry  organic  mat- 
ter which  on  February  7,  1911  amounted  to  .82  per  cent  of  the 
original  weight  of  the  corn.  A slow  evolution  of  carbon  dioxide, 
throughout  the  whole  period,  indicates  that  this  loss  is  the  result 
of  oxidation  and  it  is  believed  that  the  oxidation  is  incident  to 
respiration  of  the  living  cells  of  the  embryo.  If  the  loss  is  due 
to  oxidation,  about  60  per  cent  of  it,  or  nearly  .5  per  cent  of  the 
corn  at  the  beginning  of  the  test  is  water,  some  of  which  is  still 
present  in  the  grain.  It  is  believed  that  this  constant  produc- 
tion of  water,  small  as  it  is,  is  essential  fo,r  maintaining  the 
vitality  of  the  embryo,  and  that  it  will  continue  to  be  formed  so 
long  as  the  seeds  are  viable. 

The  total  metabolic  water  produced  in  the  corn  during  the  3% 
years  exposure  to  dry  air  is  larger  than  is  indicated  by  the  pre- 
ceding results,  since  the  oil  contained  in  corn  is  a semi-drying  oil 
which  absorbs  oxygen  directly  from  the  air  without  liberating 
an  equivalent  weight  of  carbon  dioxide.  In  consequence  of  this, 
the  dry  matter  of  the  grain  is  increased  by  an  amount  equal  to 
the  oxygen  absorbed  and  a correction  equal  to  this  should  be 
added  to  the  apparent  loss  of  water.  This  .reaction  takes  place 
slowly  at  ordinary  temperatures,  but  quite  rapidly  when  the 
temperature  is  raised. 

A sample  of  commercial  corn  oil  was  dried  by  contact  with 
anhydrous  calcium  chloride  and  afterwards  exposed,  in  a thin 
layer,  to  air  at  a temperature  of  approximately  97°.  During 
the  first  twenty-four  hours,  this  oil  gained  4.58  per  cent  in 
weight,  and  after  forty-eight  hours  the  gain  was  4.63  per  cent. 
No  further  increase  was  noted  after  longer  exposure.  The  oxi- 
dized oil  was  much  less  soluble  in  ether  than  the  fresh  oil.  The 
oil  used  in  this  test  had  been  in  the  laboratory  several  years  and 
doubtless  had  already  absorbed  some  oxygen.  It  is  probable  that 
fresh  oil,  under  similar  conditions,  would  absorb  at  least  5 per 
cent  of  its  weight  of  oxygen. 

The  ether  extract  from  fresh  corn  of  the  variety  placed  in  the 
desiccator,  averages  about  5 per  cent;  that  from  the  desiccated 
corn  was  only  3.82  per  cent,  indicating  a considerable  oxidation 
of  the  fats.  No  doubt  these  fats  are  nearly  saturated  with  oxy- 
gen and  the  dry  organic  matter  of  this  corn  has  been  increased 


96 


Wisconsin  Research  Bulletin  No.  22 


from  this  cans©  by  as  much  as  .25  per  cent,  all  of  which  should 
he  added  to  the  amount  of  metabolic  water  as  indicated  by  the 
data,  the  decrease  in  total  water,  when  the  last  determina- 
tions were  made,  may  have  been  caused  by  this  change. 

Influence  of  Drying  Upon  Germination  In  a number  of 
tests,  including  more  than  100  seeds  from  the  same  lot  of 
corn,  made  at  the  beginning  of  the  experiment,  all  kernels 
germinated  with  strong  healthy  sprouts;  subsequent  tests  of 
the  seeds  exposed  to  air  dried  by  sulphuric  acid,  made  at 
each  date  when  the  corn  was  weighed,  showed  a gradual  weaken- 
ing of  the  sprouts  although  all  germinated  until  September  28, 
1909,  when  only  seventeen  out  of  twenty  seeds  germinated ; Feb- 
ruary 21,  1910,  sixteen  out  of  twenty  germinated;  May  13,  1910, 
sixteen  out  of  twenty  germinated ; September  8,  1910,  fourteen 
out  of  twenty  germinated;  May  9,  1911,  ten  out  of  twenty  germi- 

TABLE  III.  GERMINATION  OF  DESICCATED  AND  AIR  DRIED  CORN 

•(!®r«lil?ation  :orn  ^lree  vears  in  a.desiccator  over  sulphuric  acid,  compared 
with  that  of  corn  from  the  same  lot  kept  in  a cloth  sack  exposed  to  ordinary  air 
twenty  kernels  were  tested  in  each  case. 


1 

2 

3 

4 
6 

1 

2 

3 

4 

5 

6 


Desiccated  Corn 


Corn  from  Sack 


Days  in 
Germinator 


Corn  from 


Tested  be- 
tween wet 
filters 

Immersed  in 
hydrogren 
peroxide 

Tested  be- 
tween wet 
filters 

Immersed  in 
hydrogren 
peroxide 

0 

0 

0 

0 

4 

0 

12 

8 

8 

0 

18 

18 

10 

0 

20 

20 

10 

0 

same  lots 

four  months 

later 

0 

0 

0 

0 

0 

0 

12 

10 

0 

0 

20 

20 

5 

0 

0 

0 

nated;  and  September  18,  1911,  nine  seeds  out  of  twenty  germi- 
nated. At  the  last  date,  seeds  from  the  same  lot  which  had  been 
kept  in  a cloth  sack  freely  exposed  to  air  that  was  not  excessively 
dry,  all  germinated  in  a normal  manner  with  strong  sprouts. 
The  sprouts  upon  the  desiccated  seeds  were  all  feeble.  . The 
weakened  vitality  of  the  desiccated  seeds,  compared  with  those 
kept  in  the  cloth  sack,  is  more  clearly  shown  in  Table  III  in 
which  is  given  the  time  required  for  the  germination  of  both 
samples,  when  placed  between  moist  filters,  and  when  immersed 
in  hydrogen  peroxide.  Data  are  given  for  the  last  two  tests. 


Role  of  Metabolic  Water  in  Vital  Phenomena 


97 


In  all  cases  the  sprouts  upon  the.  desiccated  corn  were  very 
weak  and  were  soon  killed  by  the  growth  of  molds. 

Nearly  all  of  the  metabolic  water  formed  during  this  period 
must  have  been  set  free  in  the  embryo,  since  the  protoplasm, 
which  functions  in  respiration,  is  chiefly  located  in  this  portion 
of  a seed.  As  perfectly  dry  protoplasm  is  incapable  of  respira- 
tion, it  seems  highly  probable  that  even  the  small  amount  of 
water  formed  under  conditions  of  extreme  dryness  may  serve 
an  important  purpose  in  prolonging  the  vitality  of  seeds. 

The  rate  of  respiration  of  seeds  is,  within  certain  limits,  deter- 
mined by  the  water  content  ; with  less  than  10  per  cent  of  water 
it  proceeds  very  slowly  and  in  this  condition  of  dryness  most 
seeds  remain  alive  and  capable  of  growth  for  long  periods  with 
a very  limited  amount  of  air.  With  15  to  20  per  cent  of  water, 
respiration  is  quite  active  so  that  oxygen  is  soon  exhausted  from 
a closed  vessel  filled  with  seeds,  and  direct  respiration  is  sus- 
pended. If  this  condition  is  continued  for  an  extended  period, 
death  of  the  seed  ensues. 

It  is  a well  established  fact  that  seeds  do  not  remain  viable  for 
a long  time  when  stored  in  large  quantities  in  tight  bins.  This  is 
especially  true  of  recently  harvested  corn,  wheat  and  oats.  These 
grains  often  contain,  at  this  time,  more  than  20  per  cent  of  water 
which  condition  stimulates  respiration  to  a point  where  the  seeds 
become  warm,  damp,  and  finally  musty.  The  rise  in  tempera- 
ture is  due  chiefly  to  respiration,  the  dampness  to  the  production 
of  metabolic  water,  the  musty  condition  to  the  growth  of  molds 
and  other  fungi,  the  development  of  which  is  favored  by  warmth 
and  excessive  moisture. 

Frequent  exposure  to  air,  by  moving  from  one  bin  to  another, 
serves  to  maintain  a better  condition  in  all  grains  when  stored 
in  bulk  and  this  plan  is  resorted  to  in  all  store  houses.  The 
usual  practice  with  com  is  to  store  it,  on  the  cob,  in  well  ventilat- 
ed cribs,  so  that  air  may  circulate  freely  throughout  the  whole 
mass.  Corn  intended  for  seed  should  never  be  stored  in  bulk  in 
large  quantities,  even  on  the  ear;  the  safest  plan  is  to  reduce 
the  water  content  to,  at  most,  10  per  cent,  by  artificial  heat, 
after  which  the  ears  should  be  kept  in  small  piles  through  which 
moderately  dry  air  may  circulate  freely;  it  should  not  be  stored 
in  a damp  place,  nor  should  it  be  shelled  long  before  planting 
time.  Seed  grains  are  more  reliable  if  kept  in  medium  sized 


98 


Wisconsin  Research  Bulletin  No.  22 


cloth  sacks  so  placed  .that  air  may  circulate  freely  between 
them.  In  this  way  normal  respiration  of  the  seeds  is  assured, 
excess  of  moisture  is  removed,  and  vital  activity  greatly  pro- 
longed. 

When  grain  is  stored  in  large  quantities,  its  moisture  in- 
creases slightly,  especially  if  the  conditions  are  unfavorable  to 
evaporation.  The  gain  that  occurs  in  these  cases  has  always 
been  attributed  wholly  to  water  absorbed  from  nearly  saturated 
air.  This  factor  is  no  doubt  operative  but  it  is  probable  that 
metabolic  water  formed  by  the  respiration  of  the  seeds,  coupled 
with  a reduced  evaporation,  has  a marked  influence  upon  the  in- 
crease in  the  water  content  of  the  grain. 

It  must  constantly  be  borne  in  mind  that  seeds  as  well  as 
growing  plants  and  animals  must  at  all  times,  have  a supply 
of  free  oxygen  if  the  highest  state  of  vitality  is  to  be  maintained, 
although  the  amount  required  by  air  dried  seeds  is  quite  small. 
Even  when  seeds  are  so  dry  that  the  growth  of  molds  and  bac- 
teria is  inhibited,  respiration  still  persists  and  if  the  seeds  are 
kept  in  air  tight  vessels,  moisture  due  to  production  of  meta- 
bolic water  accumulates,  the  free  oxygen  is  replaced  by  carbon 
dioxide,  and  after  a time  the  seeds  die. 

Influence  of  Carbon  Dioxide  upon  the  Viability  of  Seeds  It 
is  probable  that  seeds,  as  well  as  growing  plants,  may  for  a 
short  time  derive  the  energy  required  for  maintaining  vital 
processes  through  intramolecular  respiration,  but  a total  sus- 
pension of  direct  respiration  always  results,  sooner  or  later, 
in  the  death  of  a seed.  The  length  of  time  which  a seed  re- 
mains viable,  when  oxygen  is  excluded,  depends  upon  the  rate 
of  respiration,  which  varies  with  the  variety  of  seed,  its  water 
content,  and  the  temperature  to  which  it  is  exposed.  Air-dried 
corn,  containing  less  than  10  per  cent  of  water,  respires  very 
slowly,  even  a!  summer  temperatures,  and  at  temperatures  below 
freezing  its  respiration  can  scarcely  be  detected;  such  corn  can 
be  kept,  for  a year  or  more,  in  a closed  vessel  without  materially 
reducing  its  germinating  power.  Germinating  corn,  containing 
about  40  per  cent  of  water,  dies  very  quickly  when  oxygen  is 
withheld,  and  corn  containing  more  than  20  per  cent  of  water 
loses  its  germinating  power  in  less  than  two  months,  if  kept  in 
an  atmosphere  of  carbon  dioxide.  At  temperatures  below  freez- 
ing the  effect  is  much  slower,  because  respiration  is  greatly  re- 


Role  of  Metabolic  Water  in  Vital  Phenomena  99 

duced.  This  principle  is  illustrated  in  the  following  tests  made 
with  co.rn  containing  various  amounts  of  water,  from  which  the 
oxygen  was  excluded  by  filling  the  containing  vessel  with  car- 
bon dioxide. 

Seeds  from  a well  cured  ear  of  yellow  dent  corn,  of  the  crop 
of  1909,  containing  about  6 per  cent  of  water,  every  kernel  of 
which  germinated  in  several  tests,  were  hermetically  sealed  in 
an  atmosphere  of  carbon  dioxide,  November  20,  1909.  The  ves- 
sel containing  thes$  seeds  was  kept  in  a drawer,  in  the  labor- 
atory, at  a temperature  averaging  about  20° C.,  (68°F.),  and  was 
not  opened  until  November  11,  1910.  At  this  time  consider* 
able  pressure  had  developed  in  the  vessel,  showing  that  intramo- 
lecular respiration  or  anaerobic  fermentation  had  taken  place. 
The  appearance  of  the  corn  had  not  changed.  Comparative 
germination  tests  were  made  of  this  corn  and  of  corn  from  the 
same  ear  that  had  been  kept  in  air.  These  trials  were  made 
by  placing  ten  kernels  from  each  lot  between  folds  of  moist 
filter  paper,  and  also  by  immersing  the  same  number  of  seeds 
in  water  containing  1 % per  cent  of  hydrogen  peroxide.  The 
results  are  given  in  Table  IV. 


TABLE  IV.  EFFECT  OF  CARBON  DIOXIDE  UPON  VIABILITY  OF  SEEDS 

Germination  of  corn  kept  365  days  in  air  compared  with  that  of  corn  kept  in  carbon 

dioxide. 


Hours  required  for 
germination 

Corn  kept  in  air. 
Kernels  germinated 

Corn  kept  in  carbon  dioxide, 
Kernels  germinated 

I;  i wet  filters 

111  H 2°2 

In  wet  filters 

In  Hs02 

24 

0 

0 

0 

0 

48 

8 

3 

0 

0 

72 

10 

10 

r 

9 

10 

10 

j_ 

Although  all  kernels  from  both  lots  finally  germinated,  it  is 
evident,  from  the  longer  time  required  for  those  kept  in  carbon 
dioxide,  that  their  germinating  power  had  been  considerably 
weakened  by  exclusion  of  oxygen. 

Another  sample  of  yellow  dent  corn,  crop  of  1910,  taken  soon 
after  it  was  husked,  was  placed  in  carbon  dioxide  as  in  the 
first  case,  November  15,  1910.  This  corn  contained  29.66  per 
cent  of  water,  at  the  beginning,  and  in  germinating  tests  made 
at  this  time,  80  per  cent  germinated  in  contact  with  wet  filter 


100 


Wisconsin  Research  Bulletin  No.  22 


paper,  and  90  per  cent  germinated  when  immersed  in  hydrogen 
peroxide.  During  the  first  twenty-four  hours,  there  was  a mark- 
ed decrease  in  the  pressure  of  gas  within  the  vessel  indicating 
an  absorption  of  carbon  dioxide  by  the  corn;  after  this  time 
the  pressure  increased  quite  rapidly  because  of  carbon  dioxide 
liberated  by  intramolecular  respiration  and  during  the  next 
thirty  days  considerable  gas  was  evolved.  From  this  time  on 
the  pressure  remained  practically  constant,  indicating  that  re- 
spiration had  ceased  and  that  the  seeds  were  dead.  On  Jan- 
uary 12,  1911,  the  corn  was  examined  and  germination  tests 
made.  At  this  time  the  corn  was  bright  and  apparently  sound, 
hut  had  a peculiar  acid  odor  similar  to  corn  silage.  The  water 
content  had  increased  from  29.66  per  cent  to  33.94  per  cent, 
giving  unmistakable  evidence  of  active  intramolecular  respir- 
ation or  of  anaerobic  fermentation.  None  of  the  grain  germi- 
nated, either  in  contact  with  wet  filters  or  in  solution  of  hydro- 
gen peroxide.  Kernels  from  the  same  ear  kept  in  air,  all  germi- 
nated by  both  methods,  a better  result  than  was  obtained  at  the 
beginning  with  the  uncured  corn. 

A sample  of  white  dent  corn,  crop  of  1910,  containing  20.07 
per  cent  of  water  was  placed  in  an  atmosphere  of  carbon  dioxide 
November  16,  1910.  This  corn  behaved  in  a similar  manner 
to  that  in  the  previous  test,  but  the  acid  odor,  after  an  exposure 
of  two  months,  was  not  quite  so  pronounced.  None  of  this  corn 
germinated,  while  of  the  kernels  kept  in  ai,r  every  one  germi- 
nated both  in  contact  with  wet  filters  and  in  a solution  of  hy- 
drogen peroxide. 

Another  sample  of  white  dent  corn,  crop  of  1909,  that  had 
been  kept  in  the  laboratory  exposed  to  air  one  year,  and  that 
contained  8.7  per  cent  of  water,  was  placed  in  carbon  dioxide 
November  17,  1910.  These  seeds  were  removed  and  tested  July 
26,  191],  after  an  exposure  to  carbon  dioxide  for  a little  more 
than  eight  months.  The  seeds  were  found  to  be  in  good  con- 
dition and  the  ten  tested,  germinated,  both  in  contact  with  wet 
filters  and  in  a solution  of  hydrogen  peroxide.  In  this  respect 
the  seed  was  fully  as  good  as  seed  from  the  same  ear  that  had 
been  exposed  to  air. 

During  all  of  the  above  tests  with  carbon  dioxide,  direct  re- 
spiration must  have  been  suspended,  except  so  far  as  the  small 
amount  of  oxygen  retained  in  the  tissues  of  the  seed  may  have 


Role  of  Metabolic  Water  in  Vital  Phenomena  101 


served  for  the  purpose.  The  seed  must,  therefore,  have  derived 
practically  all  of  the  energy  required  for  maintaining  itsyyital 
functions,  through  intramolecular  respiration.  Some^of  the 
products  resulting  from  intramolecular  respiration  are  injurious 
to  living  cells  when  they  are  present  in  even  moderate  amounts, 
but  these  products  are  formed  so  slowly  when  the  water  con- 
tent is  below  10  per  cent  that  no  injurious  effects  are  evident 
for  a long  time;  finally,  however,  the  accumulation  of  these 
products  is  sufficiently  large  to  cause  the  death  of  a seed,  no 
matter  how  low  its  water  content  may  be.  With  a water  con- 
tent of  20  per  cent  or  over,  intramolecular  respiration  is  so  rapid 
especially  if  the  temperature  be  favorable,  that  death  of  a seed 
results  in  a short  time. 

Injury  to  Seed  through  Storage  in  Bulk  When  grains  are 
stored  in  bulk,  the  heat  generated  by  the  slow  respiration  that 
occurs  is  not  readily  dissipated  and  the  temperature  gradually 
/'rises;  with  the  increased  temperature,  respiration,  both  direct 
and  intramolecular,  is  augmented  and  in  a short  time  oxygen 
is  practically  excluded,  so  that  only  intramolecular  respiration 
is  possible.  Unless  grain  is  quite  dry,  (it  should  not  contain 
more  than  10  per  cent  of  water),  storage  in  bulk  without  fre- 
quent aeration,  to  reduce  temperature  and  to  remove  the  ac- 
cumulated carbon  dioxide  and  water,  is  sure  to  result  in  damage. 
Naturally,  greater  injury  occurs,  under  such  conditions,  near 
the  top  of  a bin  where  some  oxygen  enters  by  diffusion,  since 
this  permits  some  direct  respiration  of  the  grain  to  take  place, 
and  also  allows  molds  and  other  destructive  organisms  to 
develop ; both  of  which  contribute  to  a more  rapid  rise  in  tem- 
perature. 

Experiments  made  with  large  quantities  of  corn  by  J.  W.  T. 
Duvel2  to  determine  the  deterioration  of  corn  in  storage,  and  lat- 
er, in  cooperation  with  Laurel  Duval 3 to  determine  the  shrink- 
age of  corn  in  storage,  confirm  the  above  results  and  conclusions 
in  every  respect. 

In  the  later  test,  28,000  pounds  of  shelled  corn  with  an  average 
water  content  of  18.8  per  cent  and  an  average  germination  of 
89.6  per  cent  were  placed  in  the  wooden  hopper  of  a 30,000 
pound  elevator  scale  and  the  weights  and  temperatures  observ- 


2 Cir.  43 — Bur.  Plant  Industry.  U.  S.  Dept,  of  Agr. 

3 Cir.  81 — Bur.  Plant  Industry.  U.  S.  Dept,  of  Agr. 


102  Wisconsin  Research  Bulletin  No.  22 

ed  at  intervals.  The  experiment  was  begun  January  5,  1910,  at 
which  time  the  average  temperature  of  the  corn  was  20°F. 
Fifty  days  later,  February  24,  the  total  loss  in  weight  was  30 
pounds.  April  8,  the  total  loss  amounted  to  60  pounds,  a little 
more  than  .2  per  cent  of  the  initial  weight,  and  the  average 
temperature  was  46.3°F.  April  21  the  total  loss  was  107.5 
pounds  and  the  average  temperature  was  69.5°F.  The  highest 
temperature  at  any  point  in  the  corn  at  this  time  was  87°F., 
near  the  top  where  it  was  exposed  to  air,  and  the  lowest  tempera- 
ture was  51  °F.  near  the  bottom  where  it  was  protected  from 
air  by  the  corn  above.  Up  to  this  time  the  corn  was  in  good  con- 
dition, but  after  this  there  was  a marked  increase  in  tempera- 
ture and  a decided  falling  off  in  quality.  The  maximum  tem- 
perature May  2 was  138°F.,  nea,r  the  top,  May  12  133°F.  and 
May  14,  at  the  same  point,  119  °F. 

The  high  maximum  temperature  must  be  attributed  chiefly 
to  the  accumulation  of  heat  generated  by  direct  respiration  of 
the  cells  of  the  embryos,  although  fungus  organisms  undoubt- 
edly contributed  something  towards  it.  The  decrease  in  tem- 
perature in  the  last  few  days,  appears  to  be  due  to  the  death  of 
the  cells  of  the  grain,  and  a consequent  reduction  in  the  rate 
of  direct  respiration  of  the  corn,  since  there  is  no  reason  for  be- 
lieving that  the  growth  of  fungi  would  diminish,  so  long  as  an 
abundance  of  suitable  nutrients  was  available.  The  active  cells 
of  the  grain  are  killed  by  the  exclusion  of  oxygen  and  the  high 
temperature,  rather  than  by  direct  attack  of  lower  organisms, 
since  living  cells  are  extremely  resistant  to  such  organisms  as 
occur  in  grain.  The  relatively  low  temperature  at  the  bottom 
of  the  bin  is  due  to  a practical  suspension  of  direct  respiration 
of  the  corn  and  to  the  inability  of  other  organisms  to  develop  in 
the  absence  of  oxygen. 

The  effects  of  respiration ‘are  also  shown  by  the  loss  of  weight 
during  the  test.  So  long  as  the  temperature  remained  near  the 
freezing  point  the  rate  of  respiration  was  very  low  and  the  loss 
in  weight  insignificant,  being  less  than  .4  per  cent  for  the  first 
106  days.  With  the  temperature  above  50°F.,  however,  respi- 
ration was  enormously  increased,  causing  a sharp  rise  in  tem- 
perature, since  radiation  was  necessarily  slow  from  the  large 
mass  of  grain. 

May  14,  the  corn  was  run  out  of  the  hopper  and  elevated 


Role  of  Metabolic  Water  in  Vital  Phenomena  103 

three  times  thus  giving  it  a thorough  aeration  and  reducing  the 
average  temperature  to  55°F.,  practically  the  same  as  the  air. 
The  exposure  to  air,  and  the  mixing  of  the  live  with  the  dead 
kernels  started  active  respiration  again  throughout  the  whole 
mass  and  by  June  1,  when  the  experiment  was  terminated,  the 
corn  was  again  hot.  The  total  loss  in  weight,  during  the  147 
days  of  storage,  including  the  loss  incurred  when  the  grain  was 
aerated,  was  1970  pounds,  a little  more  than  7 per  cent.  The 
average  water  content  at  the  end  was  14.7  per  cent  and  the  aver- 
age germination  was  1 per  cent. 

There  were  at  the  beginning  of  the  test,  22,736  pounds  of  dry 
matter  and  at  the  end  22,204  pounds,  a loss  of  532  pounds. 
There  was  a direct  loss  of  1438  pounds  of  water,  or  over  5.1  per 
cent,  of  the  initial  weight,  of  the  corn.  In  addition  to  this  there 
must  also  have  been  lost  an  amount  of  water  equivalent  to  the 
metabolic  water  arising  from  the  total  oxidation  of  the  dry 
organic  matter  that  had  disappeared.  Since  sugars  and  fats 
contained  in  the  embryo  of  seeds  are  the  first  substances  to  be 
oxidized  in  respiration,  and  since  the  total  oxidation  of  100 
parts  of  these  substances  results  in  the  production  of  from  60  to 
more  than  100  parts  of  water,  it  is  safe  to  assume  that  at  least 
60  per  cent  of  the  dry  matter  lost  in  these  tests  or  319.2  pounds 
has  reappeared  as  water.  This  amounts  to  a little  more  than 
1.1  per  cent  of  the  total  initial  weight.  The  “sour”  condition 
and  decreased  viability  of  the  corn  at  the  end  of  the  test  are 
sure  indications  that  intramolecular  respiration  had  taken  place 
or  that  anaerobic  organisms  were  present  in  large  numbers,  since 
an  acid  condition  does  not  result  from  the  direct  respiration  of 
seeds. 

Enzymes  of  Seeds  Nea,rly  all  of  the  stored  food  materials 
of  seeds,  (starch,  proteins,  fats,  etc.),  are  insoluble  and  unavail- 
able for  nourishing  the  embryo,  until  acted  upon  by  certain 
specific  ferments,  (diastase,  proteolytic  ferments,  lipase,  etc.), 
which  change  them  into  soluble  and  diffusible  products.  These 
enzymes  appear  to  be  wholly  absent  from  immature  seed4.  It 
they  were  present  at  this  stage,  even  in  small  amount,  all  of 
the  reserve  food  would  be  changed  into  a soluble  form  and 
either  lost  to  the  seed  by  diffusion  to  other  parts  of  the  plant, 

4 See  Experiment  described  in  paragraph  devoted  to  germination  of 
immature  seed  on  page  129. 


104 


Wisconsin  Research  Bulletin  No.  22 


or  cause  the  seed  to  germinate  prematurely,  since  moisture  and 
temperature  conditions  are  favorable  for  these  purposes  at  the 
tune  when  such  substances  are  deposited.  So  long  as  a seed  is 
attached  to  the  parent  plant  by  living  tissue  and  receives  its  sup- 
ply of  food  material  therefrom,  its  condition,  is  analogous  to 
that  of  the  fetus  of  an  animal  which  receives  its  nutrients  and 
respires  indirectly  through  the  parent.  During  this  period 
there  is  no  need  for  the  action  of  such  enzymes,  and  indeed  their 
presence  would  be  positively  detrimental  because  the  reserve 
food  material,  upon  which  the  support  of  the  future  plant  de- 
pends for  its  early  development,  would  be  dissipated. 

As  a seed  matures,  direct  connection  with  the  vascular  system 
( f the  mother  plant  is  broken  and  the  seed  soon  dries  to  a point 
where  the  action  of  ferments  is  practically  suspended.  At  this 
stage,  direct  respiration,  which  lias  previously  been  prevented 
by  the  succulent  tissues,  (either  husks  or  fleshy  fruits,  which 
surround  the  seed)  is  also  established.  The  rate  of  respiration 
is  at  first  very  slow  for  the  reason  that  there  is  but  little  easily 
oxidizable  material  present  in  the  embryo  and  also  because  free 
oxygen  is  still  to  a considerable  degree  excluded  by  the  en- 
velopes that  surround  the  seed. 

The  protoplasmic  activity  of  a seed  is  greatly  stimulated  by 
direct  respiration  and  results  in  the  production  of  small  quanti- 
ties of  the  specific  enzymes,  (diastase  etc.),  required  to  render 
the  reserve  food  material  available  for  the  embryo.  If  the  seed 
is  moderately  dry,  containing  less  than  10  per  cent  of  water, 
or  if  the  temperature  is  near  the  freezing  point  or  below,  these 
changes  are  very  slow,  the  seed  remaining  practically  dormant 
for  a long  period,  but  capable  of  .resuming  its  activity  when 
favorable  conditions  are  supplied.  With  a moisture  content  of 
over  30  per  cent,  and  a favorable  temperature,  the  rate  of  respi- 
ration is  enormously  increased  and  as  a consequence  diastase  and 
proteolytic  ferments  are  produced  in  sufficient  quantity  to  ra- 
pidly convert  the  stored  starch  and  proteins  into  soluble  and 
diffusible  forms.  The  abundance  of  easily  assimilated  food 
thus  supplied  stimulates  the  embryo,  to  still  greater  activity  in 
this  direction,  and  finally  causes  the  seed  to  germinate.  This 
principle  is  utilized  in  the  manufacture  of  malt  from  barley 
grains  which  contain  very  little  diastase  until  germinated  under 
proper  conditions,  but  afterwards  are  rich  in  this  enzyme,  while 
the  starch,  at  first  present,  has  wholly  disappeared.  . 


Hole  of  Metabolic  Water  in  Vital  Phenomena  105 


The  embryo  of  a seed  will  starve  and  its  respiration  cease  if 
suitable  food  is  withheld  for  an  extended  period,  the  length  of 
which  varies  with  the  variety  of  seed  and  its  environment. 
This  sometimes  happens  in  old  dry  seeds  in  which  the  inverting 
enzyme  has  either  become  dormant  through  long  continued  in- 
activity or  has  been  destroyed  by  an  accumulation  of  waste  pro- 
ducts in  the  seed.  It  seems  that  a failure  of  specific  enzymes  to 
act  may  be  one  of  the  chief  factors  which  limits  the  period  of 
viability  of  seeds. 

This  view  is  in  a measure  confirmed  by  the  behavior  of  some 
varieties  of  seeds,  which  have  lost  much  of  their  germinating 
power  through  age,  when  soaked  in  solutions  of  enzymes  capa- 
ble of  rendering  the  reserve  food  material  of  the  seed  soluble 
and  available  to  the  embryo.  F.  A.  Waugh  5 obtained  great  im- 
provement in  the  germination  of  old  seeds  when  they  were 
treated  with  solutions  of  various  enzymes.  Table  V shows  some 
of  his  results. 


TABLE  V.  INFLUENCE  OF  ENZYMES  UPON  GERMINATION 


Description  of  seeds 

Solution  em- 
ployed 

Per  cent 
germination 

Tomato,  12  years  old 

W a.t.er . _ . 

12 

Tomato,  12  years  old 

Dia.stasp.  . 

85 

Tomato,  12  years  old 

W a.tpr 

34 

Tomato,  12  years  old 

Pia.*t,a,*p  . 

70 

Tomato,  12  years  old 

W a ter 

14 

Tomato,  12  years  old 

Dia.sta.sp. 

24 

Tomato,  5 years  old 

Wa.tpr  . 

36 

Tomato,  5 years  old 

Diastase 

46 

Cucumber,  5 years  old 

W a, ter 

44 

Cucumber,  5 years  old 

Diastase 

54 

Radish,  G years  old 

W ater . . . 

46 

Radish,  6 years  old 

Diastase 

66 

Many  other  experiments  with  different  seeds  and  a variety 
of  enzymes  most  of  which  increased  the  germination,  are  de- 
scribed in  Waugh’s  paper.  In  general,  diastase  improved  ger- 
mination more  than  other  enzymes  employed. 

No  doubt  each  variety  of  seed  is  supplied  with  particular 
enzymes,  or  combinations  of  enzymes,  which  serve  its  purpose 
better  than  is  possible  with  any  artificial  preparation.  It  would 


5 Vt.  Agr.  Exp.  Sta.  Rpt.,  189G.  p.  106. 


106 


Wisconsin  Research  Bulletin  No.  22 


seem  that  diastase  should  be  especially  adapted  to  starchy  seeds 
like  corn,  wheat,  barley  etc.  while  proteolytic  enzymes  such  as 
pepsin  and  trypsin  would  be  more  beneficial  to  leguminous  seeds 
and  that  lipase  would  serve  the  purpose  best  for  seeds  rich  in  fat. 

A similar  experiment  to  the  above  was  conducted  at  this  Sta- 
tion in  November  1909,  with  corn,  less  than  50  per  cent  of  which 
germinated  when  the  seeds  were  soaked  in  water  only.  Seeds 
from  the  same  lot  and  treated  in  the  same  manner  as  the  checks, 
except  that  the  water  in  which  they  were  soaked  contained  com- 
mercial diastase,  all  germinated.  The  maximum  growth  was 
about  the  same  in  each  lot,  but  the  growth  of  the  seeds  soaked 
in  diastase  was  very  uniform  while  that  of  the  water  lot  varied 
greatly.  The  increased  vitality  of  the  diastase  lot  was  very 
noticeable. 

Seeds  from  the  same  lot  that  were  soaked  fifteen  hours  in  a 
3 per  cent  solution  of  glucose  instead  of  water,  all  germinated, 
thus  confirming  the  view  that  lack  of  suitable  food  was  the 
chief  reason  why  the  untreated  seed  germinated  poorly.  In 
this  case,  there  was  probably  a lack  of  a starch-inverting  enzyme 
in  the  seed  since  equally  good  results  were  obtained  when  either 
diastase  or  glucose  was  supplied;  either  would  stimulate  respi- 
ration, which  once  established,  results  in  the  production  of  the 
enzymes  essential  to  the  conversion  of  the  stored  nutrients  into 
an  available  form. 

It  has  been  suggested  that  even  immature  seeds  may  contain 
all  of  the  specific  enzymes  required  for  the  changing  of  nutri- 
ents into  forms  that  can  be  utilized  by  the  embryo  and  that  their 
action  is  prevented  by  certain  anti-ferments  which  are  present 
at  this  stage  of  growth  but  which  are  destroyed  with  the  drying 
of  the  seed  at  maturity.  It  seems  to  me  far  more  probable  that 
these  enzymes  are  wholly  absent  from  a seed  so  long  as  it  re- 
ceives its  nutriment  from  the  parent  plant  and  are  only  pro- 
duced when  direct  and  independent  respiration  of  the  embryo 
is  established.  It  is  difficult  to  explain  on  any  other  basis  why 
the  amount  of  these  enzymes  increases  at  such  a rapid  rate  with 
the  increased  respiration  that  occurs  during  germination. 

Whatever  their  source,  the  function  of  such  enzymes  is  un- 
questionably to  convert  the  reserve  nutrients  of  the  seed  into 
available  forms  and  thus  maintain  the  life  of  the  embryo  until 
it  reaches  a state  of  development  in  which  photosynthesis  can 


Role  of  Metabolic  Water  in  Vital  Phenomena  107 

take  place.  It  is  even  possible  to  continue  growth  for  consider- 
able periods,  after  the  reserve  material  of  the  seed  is  exhausted, 
without  photosynthesis,  if  proper  nutrients  are  supplied  to  the 
embryo  from  external  sources.  This  is  shown  by  the  experi- 
ments of  Maze6  who  succeeded  in  growing  vetches  in  darkness  to 
a considerable  size  in  sterilized  nutrient  solutions  containing 
glucose.  Some  of  his  results  are  given  in  Table  VI. 
table  vi.  assimilation  of  carbohydrates  by  vetches  in 

DARKNESS 


1. 

2. 

3. 

4. 


6(a) 


. Per  cent 

Days  of  glucose 

experiment  supplied 


Dry  weight 
of  seed, 
M,rs. 


Dry  weight 
of  plant. 
Mgs. 


50 

39 

92 

92 

53 

53 


1 202.8 

2 202.8 

4 202.8 

6 202.8 

0 202.8 

0 202.8 


269.0 
276.7 
838.2 

710.0 
161.6 
133.4 


(a)  No  nitrogen  supplied. 

The  plants  grown  in  the  glucose  solutions  were  much  more 
vigorous  than  those  in  the  check.  Their  tap  roots  were  strong 
and  well  branched,  and  the  stems  attained  a much  greater  length, 
amounting  to  as  much  as  1.3  meters,  with  branches  extending 
1.65  meters. 

If  the  embryos  be  carefully  removed  from  soaked  grains  of 
corn  and  placed  in  contact  with  starch  paste,  they  will  sprout 
and  grow  as  freely  as  do  those  left  in  the  unmutilated  seed. 

These  results  indicate  that  the  presence  of  specific  enzymes  is 
not  essential  to  the  growth  of  an  embryo,  if  food  material  m 
suitable  form  is  supplied  from  external  sources. 

Distribution  of  Absorbed  Water  in  Seed  When  air  dried 
seeds  are  immersed  in  water,  they  will  in  a few  hours  absorb 
more  than  their  weight  of  water,  and  will  increase  considerably 
in  volume.  If  the  water  in  which  the  seeds  are  soaked  has  been 
freed  from  air  by  boiling,  the  seeds  will  become  saturated  with- 
out any  signs  of  germination.  The  rate  at  which  air  dried  seeds 
of  corn  absorb  water,  when  immersed  in  it,  and  the  distribution 
of  this  absorbed  water  between  the  embryo  and  the  starchy  por- 
tions is  shown  in  Table  VII.  It  seems  likely  that  similar  rela- 
tions hold  for  all  varieties  of  seeds. 

eCompt.  Rend.,  1899,  pp.  185-187.  Abstracted  in  Exp.  Sta.  Record, 
Vol.  XI,  1899-1900  p.  317. 


108 


Wisconsin  Research  Bulletin 


No.  22 


it  will  be  seen  that  the  water  taken  up  by  the  seed  is  not 
uniformly  distributed  between  the  embryo  and  the  starchy  por- 
tion and  also  that  the  embryo  absorbs  water  more  rapidly  than 
other  parts  during  the  first  24  hours,  after  which*  the  rate  is 


TABLE  VII. 


EFFECT  OF  SOAKING  CORN  ON  ITS  WATER  CONTENT 


Amount  of  water  in  the  embryo  and  starchy  portion  of  air  dried  corn  compared  with 
that  in  corn  soaked  for  different  periods  in  boiled  water. 


Air  dried 


Hours  soaked  in  water 
2.5 


20 

24 

28 

48....... 

48 

72 

100 


Yellow  Dent 


Percent  water 
in  embryo 


5.79 

5.59 

0.39 


31.20 
47  A9 


Percent  water 
in  starchy 
portion. 


White  Dent 


7.07 

7.08 
8.57 


19.67 

30!30' 


Percent  watei 
in  embryo 


5.83 

5.65 


23.50 

'si! 62’ 


Percent  water 
in  starchy 
portion 


8.99 

7.11 


17.82 
3i  .57- 


54.41 

53.87 

56.11 


35.48 

35.43 

38.54 


52.86 

58.17 


58.78 

59.55 


33.15 

35.27 


37  59 
39.40 


practically  the  same  in  all  portions.  The  more  rapid  absorption 
by  the  embryo  at  first  appears  to  be  chiefly  due  to  its  peculiar 
structure  and  its  location  near  the  surface  rather  than  to  a 
more  hygroscopic  nature  of  its  substance.  The  relatively  low 
specific  gravity  of  the  embryo  compared  with  that  of  whole 
kernels,  shown  in  Table  VIII  made  upon  three  separate  sam- 
ples of  corn,  suggests  a more  porous  structure  of  the  embryo. 


TABLE  Till.  SPECIFIC  GRAVITY  OF  GRAINS  OF  CORN 


Sample 

Specific  gravity 

Specific  gravity 

of  whole  kernels 

without  embryos 

1..... 

1.2931 

1.2940 

1.2996 

1.3449 

3!!!.!!!!!!!! 

— 

1.3135 

1.3039 

Tile  difference  between  the  specific  gravity  of  the  embryo  and 
that  of  the  starchy  portion  of  the  kernel  is  really  much  greater 
than  these  figures  indicate,  since  the  embryo  comprises  only 
about  one  tenth  of  the  whole  kernel.  No  doubt  the  low  specific 
gravity  of  the  embryo  is  partly  caused  by  the  large  proportion 


Kole  of  Metabolic  Water  in  Vital  Phenomena  109 


of  fat  which  it  contains  but  it  is  probably  due  mostly  to  a more 
porous  structure.  Tf  the  substance  of  the  embryo  were  more 
hygroscopic  than  other  parts  of  the  kernel,  it  should  contain 
relatively  more  water  than  the  starchy  portion  in  air  dried  seeds, 
a condition  which  has  not  been  observed  in  any  determinations 
made  upon  corn.  (See  Table  VII).  Moreover  the  embryos, 
after  being  removed  from  the  seed  and  dried,  have  in  no  case 
taken  up  water,  when  exposed  to  air,  as  rapidly  as  the  starchy 
portions  of  the  seed,  but  when  immersed  in  water  both  parts 
behave  as  in  the  fresh  seed. 

The  low  percentage  of  water  in  the  embryo  of  an  air  dried 
kernel  of  corn  is  doubtless  due  to  the  substance  composing  the 
embryo  having,  on  the  whole,  a weaker  affinity  for  water  than 
does  starch;  the  large  proportion  of  fat,  which  has  practically 
no  affinity  for  water,  in  the  tissues  of  the  embryo  is  an  important 
factor  in  determining  this  condition.  The  more  rapid  absorp- 
tion of  water  when  a seed  is  immersed  in  water  is  probably  due 
to  unoccupied  spaces  in  the  embryo  and  in  the  tissues  immediate- 
ly surrounding  it,  as  these  spaces  are  quickly  filled  with  water. 
It  is  analogous  to  the  absorption  of  water  by  a sponge,  the  sub- 
stance of  which  may  not  in  itself  have  a strong  attraction  for 
water.  There  is  however  abundant  evidence  to  show  that  starch, 
which  constitutes  a large  proportion  of  a kernel  of  corn,  as  well 
as  of  many  other  seeds,  forms  feeble  molecular  combinations 
w$fh  water  analogous  to  those  in  crystals  containing  water  of 
crystallization,  and  no  doubt  the  same  is  true  of  the  protein 
substances  found  in  seeds.  The  water  content  of  starch  and 
corn  that  have  been  exposed  to  moist  air  is  practically  the  same, 
indicating  that  the  nature  of  the  combination  is  the  same  in  both 
cases. 

Changes  Occurring  During  Germination 

If  the  water  content  of  a mature  and  air  dried  seed  is  raised 
to  over  30  per  cent  and  the  seed  is  afterwards  exposed  to  air  or 
to  free  oxygen,  at  a suitable  temperature,  the  vital  activity  of 
the  embryo  is  enormously  increased.  This  activity  is  manifested 
by  a rapid  absorption  of  oxygen,  a corresponding  evolution  of 
carbon  dioxide,  an  increase  in  the  water  content  of  the 
embryo,  and  a loss  in  the  total  dry  matter  of  the  seed.  In  a 
short  time,  which  varies  with  the  variety  of  seed,  new  cells  are 


110  Wisconsin  Research  Bulletin  No.  22 

formed  and  a sprout  is  pushed  out  from  the  embryo  which  finally 
develops  into  the  root  of  the  future  plant.  Shortly  after  the 
root  is  started,  another  shoot  is  formed  which  is  destined  to  be- 
come the  stem.  This  production  of  new  tissue  of  a definite  and 
,*  characteristic  form,  from  material  within  a seed,  is  designated 
by  the  term  “ germinationT'S 

Conditions  Affecting  Germination 

A plentiful  supply  of  water,  a favorable  temperature,  and 
the  presence  of  free  oxygen  are  essential  for  the  germination  of 
all  seeds. 

W ater  The  stored  nutrients  of  a seed  are  mostly  insoluble 

and  consequently  unavailable  for  the  embryo  until  they  are  ren- 
dered soluble  by  combination  with  the  elements  of  water.  This 
change  is  effected  by  specific  ferments,  the  production  of  which 
is  dependent  upon  the  direct  respiration  of  the  active  cells  of 
the  embryo;  it  takes  place  very  slowly  except  in  the  presence 
of  considerable  water.  With  corn  the  change  is  extremely  slow 
when  the  water  content  is  less  than  20  per  cent  but  the  rate 
increases  rapidly  when  the  water  content  is  raised  above  this; 
no  sprouts  appear,  however,  until  the  water  amounts  to  about 
25  per  cent  of  the  total  weight  of  the  seeds;  above  this  point 
hydrolysis  of  stored  nutrients  proceeds  with  sufficient  rapidity 
to  supply  all  needs  of  the  active  cells  of  the  embryo.  Not  only 
is  water  needed  for  this  purpose  but  there  must  be  provided, 
m addition,  a sufficient  quantity  of  water  to  dissolve  the  pro- 
ducts formed  and  to  transfer  them  by  osmosis  to  the  cells  where 
respiration  occurs.  On  the  other  hand,  any  excess  of  water 
above  that  which  a seed  can  absorb  is  a disadvantage,  since  it 
removes  soluble  nutrients  "which  are  needed  for  the  proper  nour- 
ishment of  the  growing  cells  and  also  interferes  with  respiration 
by  excluding  oxygen.  In  consequence  of  this,  a large  surplus 
of  water  greatly  weakens  germination  and  may  wholly  prevent 
it.  . 

Temperature  The  range  of  temperature  within  which  ger- 
mination may  take  place,  depends  upon  the  variety  and  condi- 
tion of  seed.  Some  seeds  germinate  in  contact  with  melting  ice 
at  a.  temperature  approximating  0°C.,  as  is  the  case  with  wheat, 
but  the  seeds  of  most  cultivated  plants  fail  to  germinate  below 
4°  C.  (39°F.)  The  optimum  temperature  for  these  plants  is 


Role  of  Metabolic  Water  in  Vital  Phenomena  111 


about  30°  C.  (86°  F),  and  germination  is  unsatisfactory  above 
40°  C.  (104°  F.).  The  time  required  for  germination  is  greater 
as  the  temperature  is  reduced  below,  or  raised  above  the 

optimum. 

Oxygen  Although  intramolecular  respiration  may  occur 
with  seed  containing  more  than  10  per  cent  of  water,  when  free 
oxygen  is  excluded,  germination  never  takes  place  except  under 
conditions  that  admit  of  direct  respiration.  Free  oxygen  is, 
therefore,  essential  although  its  presence  in  the  gaseous  state 
is  not  always  necessary.  Some  seeds,  when  immersed  in  water 
are  able  to  utilize  dissolved  oxygen,  but  none  except  those  from 
water  plants  can  make  an  extended  growth  under  these  con- 
ditions. 

About  twenty  varieties  of  farm  and  garden  seeds  have  been 
tested  and  all  found  to  decompose  hydrogen  peroxide  rapidly. 
When  immersed  in  solutions  containing  this  reagent,  they  were 
in  most  cases  able  to  use  the  liberated  oxygen  for  direct  respira- 
tion; and  germinated  under  these  conditions  as  quickly  as  if 
exposed  to  air. 

Germination  Tests  in  Hydrogen  Peroxide 

The  moisture,  temperature  and  atmospheric  conditions  favor- 
able to  germination  are  also  ideal  for  the  growth  of  molds  and 
other  fungi  that  feed  upon  the  soluble  nutrients  of  a seed,  and 
when  such  organisms  gain  the  ascendency  they  are  sure  to  in- 
terfere seriously  with  the  normal  progress  of  ge,rmination.  The 
most  common  organisms  of  this  kind  do  not  directly  attack  the 
living  cells,  but  by  withdrawal  of  nutrients  and  production  of 
poisonous  excretions  cause  a feeble  growth  and,  if  present  in 
large  numbers,  especially  in  the  early  stages  of  germination,  be- 
fore photosynthesis  is  established,  are  sure  to  result  in  the 
death  of  the  embryo. 

When  grown  in  soil  under  natural  conditions,  most  healthy 
seeds  overcome  these  attacks,  but  when  grown  in  the  laboratory 
between  wet  cloths  or  filters  o,r  even  in  moist  air,  molds  and 
mildews  are  almost  certain  to  appear  and  vitiate  results,  if  an 
experiment  is  extended  beyond  three  or  four.  days.  Attempts 
have  been  made  during  this  investigation  to  avoid  the  growth 
of  such  organisms  by  using  sterilized  apparatus  but  even  when 
this  was  done  the  spores  adhering  to  the  seeds  were  almost  al- 
ways sufficient  to  infect  them  and  cause  trouble  before  an  ex- 


112 


Wisconsin  Research  Bulletin  No.  22 


periment  was  ended.  No  better  success  was  attained  when 
the  seeds  were  subjected  t9  antiseptics,  as  germination  was  al- 
ways weakened  and  often  entirely  prevented,  if  treatment  of 
the  seed  was  sufficiently  prolonged  to  destroy  spores. 

Among  other  reagents  employed  for  this  purpose  was  hydro- 
gen peroxide,  in  which  kernels  of  corn  were  immersed  for  pe- 
riods ranging  from  twenty-four  tp  seventy-two  hours.  The 
strength  of  the  hydrogen  peroxide  solution  used  varied  from  14 
to  3 per  cent.  Seeds  removed  from  any  of  these  solutions,  and 
exposed  to  air,  germinated  as  well,  apparently,  as  untreated 
seed  and,  if  exposed  in  sterile  vessels,  were  usually  free  from 
mold.  Seeds  kept  in  the  solution  from  forty-eight  to  seventy- 
two  hours  germinated  in  the  solution,  with  no  direct  contact 
with  air,  the  oxygen  required  for  respiration  being  derived 
from  the  reagent.  When  corn  is  germinated  in  this  way,  the 
sprouts  grow  to  about  ^4  inch  in  length,  after  which  the  tip 
begins  to  curl  and  no  further  growth  occurs,  but  if  the  seeds 
be  removed  from  the  hydrogen  -peroxide,  even  at  this  stage, 
and  exposed  to  light,  photosynthesis  begins  and  growth  is  re- 
newed. 

Good  results  were  obtained,  with  corn,  with  all  strengths  of 
solutions  tried,  the  most  favorable  being  with  a iy2  per  cent 
solution  prepared  by  diluting  the  commercial  3 per  cent  solu- 
tion with  an  equal  volume  of  water. 

The  tests  were  made  by  placing  a few  kernels  of  corn,  or 
other  seeds,  in  a test  tube  or  small  Erlenmeyer  flask  with  four 
or  five  times  their  volume  of  the  solution  of  hydrogen  peroxide. 
The  tube  or  flask  was  partially  closed  by  inserting  a small  test 
tube,  containing  water,  in  the  mouth;  this  tube  should  reach 
below  the  surface  of  the  liquid  in  the  tube  containing  the  seed. 
This  arrangement  permits  the  escape  of  oxygen  as  it  forms  and 
keeps  the  seed  beneath  the  surface  of  the  liquid.  Some  device 
of  this  kind  is  necessary  as  bubbles  of  oxygen  adhere  to  the 
seeds  causing  them  to  float. 

The  best  results  have  been  obtained  when  the  volume  of  the 
hydrogen  peroxide  solution  was  less  than  ten  times  that  of  the 
seeds  tested.  This  is  probably  % due  to  the  removal  of  a large 
proportion  of  the  soluble  nutrients  of  the  seed  by  the  excess  of 
water.  It  must  be  borne  in  mind  that  a sufficient  supply  of 
soluble  organic  nutrients,  of  the  right  kind  to  maintain  respi- 
ration, is  as  essential  to  germination  as  is  a supply  of  oxygen. 


Hole  of  Metabolic  Water  in  Vital  Phenomena  113 


A very  satisfactory  method  of  making  germination  tests  is 
to  place  the  seeds  between  filter  papers  that  are  afterwards 
moistened  with  a 1 y2  per  cent  solution  of  hydrogen  peroxide. 
In  this  way  a large  excess  of  the  reagent  is -avoided  and  growth 
of  parasitic  organisms  prevented.  It  is  well  in  this  case  to 
renew  the  solution  after  twenty-four  hours,  the  surplus  liquid 
being  poured  off  or  absorbed  by  dry  filter  paper.  In  general, 
small  seeds  such  as  tobacco,  timothy,  clover,  etc.,  have  not  ger- 
minated as  readily  with  hydrogen  peroxide  as  when  water  only 
was  employed.  Good  results  have  been  obtained  with  corn, 
wheat,  rye,'  barley,  buckwheat,  peas  and  beans  either  when  im- 
mersed in  the  reagent  or  when  placed  btween  filter  papers  and 
moistened  with  it.  Oats  have  not  germinated  well,  by  either 
method,  unless  the  hulls  were  previously  removed ; when  this 
was  done  oats  germinated  as  well  in  hydrogen  peroxide  as 
tween  wet  filters.  It  has  also  been  noticed  that  corn  of  low 
vitality,  as  shown  by  a low  percentage  of  germination,  requires  a 
longer  time  for  germination  in  hydrogen  peroxide  than  in  water. 
This  suggests  that  the  method!  may  serve  the  jiurpose  of  dis- 
criminating between  doubtful  and  good  seed. 

It  is  noteworthy  that  immature  corn  which  failed  to  ger- 
minate in  wet  filters  germinated  perfectly  after  immersion  for 
two  weeks  in  hydrogen  peroxide. 

Distribution  of  Water  in  Germinating  Corn 

Kernels  of  corn,  that  have  been  immersed  twenty-four  hours 
or  longer  in  boiled  water  free  from  air,  do  not  germinate,  al- 
though they  absorb  sufficient  water  to  cause  germination  when 
oxygen  is  supplied.  So  long  as  oxygen  is  withheld  from  such 
seeds,  the  absorbed  water  remains  distributed  between  the  em- 
bryo and  the  starchy  portion  of  the  kernel  in  the  manner  shown 
in  Table  VII.  If,  however,  oxygen  is  supplied  freely  the  per- 
centage water  content  of  the  embryo  increases  rapidly  as  ger- 
mination proceeds  and  when  sprouts  are  formed  these  are  far 
more  succulent  than  the  embryo.  During  this  period  the  per 
cent  of  water  in  the  starchy  portion  of  the  kernel  is  but  slightly 
reduced. 

During  the  past  two  years,  a number  of  tests  have  been  made 
to  determine  these  changes  in  the  distribution  of  water  in  ker- 
nels of  corn  during  the  early  stages  of  germination.  Two  varie- 


114  Wisconsin  Research  Bulletin  No.  22 

ties  of  corn,  a white  and  a yellow  dent,  grown  upon  the  station 
grounds  in  1909  and  1910  were  used  in  these  tests.  In  each  test 
kernels  nearly  uniform  in  size  and  appearance  were  selected 
from  the  same  ear  and  soaked  for  twenty-four  hours  in  about 
ten  times  thei,r  volume  of  water  that  had  been  previously  boiled 
to  expel  air.  From  a portion  of  these  soaked  kernels,  freed 
from  adhering  water  by  contact  with  dry  filter  paper,  the  em- 
bryos were  carefully  removed  and  water  determinations  made 
separately  in  both  parts,  by  drying  in  a steam  oven  at  approxi- 
mately 97°  C.  Other  kernels,  from  the  same  lot  of  soaked 
corn,  were  exposed  to  nearly  saturated  air  without  being  in  con- 


TABLE  IX.  EFFECT  OF  GERMINATION  ON  WATER  IN  SEEDS 

Distribution  of  water  and  dry  matter  between  the  embryos  and  starchy  portions  of 
grains  of  corn,  before  and  during  the  early  stages  of  germination. 


Condition  of  corn  when 
tested 

Whole 

grain 

Embryo 

Starchy 

portion 

I.  Air  dried 

Per  cent  water. 

6.90 

5.62 

7.10 

Per  cent  dry  matter. 

93.10 

94.38 

92.90 

II . Soaked  24  hours  in 

Percent  water 

35.97 

54.24 

32.10 

boiled  water 

Per  cent  dry  matter. 

64.03 

45.76 

67.90 

III.  Soaked  grain  after 

Percent  water 

37.53 

62.13 

31.40 

24  hours  in  germinator 

Per  cent  dry  matter. 

62.47 

37.87 

68.60  • 

IV.  Soaked  grain  after 

Per  cent  water 

38.32 

65.82 

31.03 

48  hours  in  germinatorj 

Per  cent  dry  matter. 

61.68 

34.18 

68.97 

tact  with  a wet  surface.  Under  these  conditions  sprouts  grew 
to  between  % and  % inch  in  the  first  twenty-four  hours,  and  to 
about  one  inch  in  forty-eight  hours.  At  each  of  these  periods 
water  determinations  were  made  in  the  sprouted  embryos  and  in 
the  starchy  portions  of  the  kernels  in  the  same  manner  as  in  the 
soaked  seed.  Very  satisfactory  and  uniform  results  were  ob- 
tained at  these  three  stages,  but  when  the  germinated  kernels 
were  kept  in  moist  air  for  another  period  of  twenty-four  hours, 
there  was  usually  a considerable  difference  in  the  length  of 
sprouts  upon  different  kernels  and  often  molds  also  appeared, 
which  vitiated  results. 

The  average  per  cent  of  water  found  in  the  different  parts  of 
the  kernels,  as  well  as  its  distribution  between  the  embryos  and 
starchy  portions,  for  the  first  forty-eight  hours  of  germination, 
are  given  in  Tables  IX  and  IXA. 


Role  of  Metabolic  Water  in  Vital  Phenomena  115 


Water  content  of  sprouts,  roots  and  stems  after  separation 
from  the  embryo,  lias  ranged  from  84.80  per  cent  to  90.17  per 
cent,  and  has  averaged  87.75  per  cent. 

It  will  be  noted  that  the  per  cent  of  water  is  higher  in  the 
germinated  grain  than  in  the  soaked  grain,  and  also  that  the 
per  cent  of  water  has  increased  as  the  period  of  germination 
has  been  extended,  although  the  seed  has  at  no  time,  during 
this  period,  been  in  direct  contact  with  water.  The  most 
striking  change,  however  is  in  the  water  content  of  the  embryo 
which  has  been  greatly  increased  by  germination,  in  fact  the 

TABLE  IX  A.  GRAMS  WATER  IN  PARTS  OF  SEEDS 


Distribution  of  water  and  dry  matter  between  the  embryo  and  starchy  portion  of 
grains  of  corn  in  different  conditions — Calculated  for  100  grams  of  seed. 


Condition  of  corn  when  tested 

Embryo 

grams 

Starchy 

portion 

grams 

T Air  dripd 

Water 

.77 

6.13 

Dry  matter  — 

12.94 

80.16 

Total  weight . 

13.71 

86.29 

II  Soaked  24  hrs.  in  boiled  water 

Water 

9.45 

26.48 

Dry  matter 

8.02 

56.01 

Total  weight.. 

17.51 

82.49 

III.  Soaked  grain  after  24  hrs.  in  germina- 
tor 

Water 

12  34 

25.19 

Dry  matter  — 

7.51 

5*.  96 

Total  weight.. 

19.85 

80.15 

IV.  Soaked  grain  after  48  hrs.  in  germina- 

t.nr.  

Water 

13.79 

24.53 

Dry  matter 

7.15 

54.53 

Total  weight.. 

20.94 

79.06 

whole  increase  in  water  content  has  taken  place  in  this  portion 
of  the  seed,  since  the  per  cent  of  water  in  the  starchy  parts  has 
steadily  diminished  as  germination  has  advanced. 

The  increase  in  the  water  content  of  the  embryo,  and  the  de- 
crease in  the  starchy  portion,  might  be  explained  by  a direct 
transfer  of  water  from  the  starchy  parts  of  seed  to  the  em- 
bryo, were  it  not  for  the  increase  in  the  water  content  of  the 
whole  seed,  in  spite  of  a continual  loss  of  water,  through  re- 
spiration, the  most  of  which  has  been  from  the  embryo,  where 
respiration  is  most  active,  and  also  through  fixation  of  water  in 
the  hydrolysis  of  starch  and  other  stored  nutrients.  It  is  pos- 
sible that  some  water  has  been  absorbed  from  the  nearly  satu- 


116 


Wisconsin  Research  Bulletin  No.  22 


rated  air,  but  corn  containing  as  much  water  as  did  the  soaked 
kernels  in  the  above  tests  takes  up  more  water  very  slowly,  un- 
less it  is  in  contact  with  a wet  surface,  and  even  then  the  embryo 
does  not  absorb  water  much  if  any  more  rapidly  than  other 
parts  of  the  seed.  It  may  even  be  questioned  whether  sufficient 
water  is  absorbed  from  saturated  air,  by  a germinating  seed 
to  replace  that  lost  by  transpiration. 

Tests  were  made  to  determine  the  rates  at  which  corn,  con- 
taining different  per  cents  of  water,  increases  in  water  content 
when  exposed  to  saturated  air.  Only  one  variety  of  corn  a 
white  dent,  was  used  in  these  trials.  The  results  are  shown’  in 
Table  X. 


TABLE  X.  W ATER  IN  CORN  IN  SATURATED  AlR 
This  shows  the  absorption  of  water  by  corn  exposed  to  saturated  air. 


Days  exposed 

Water  content 

0 

Lot  I 
% 
8.42 

1C  A A 

Lot  II 

% 

1 

8.42 

J 0 . ‘±{t 
00  A1 

14.25 

16 

66.  VI 

OK  Cl 

20.61 

6D  .01 
OA  HA 

24.52 

0 

♦wO.uo 

26.70(a) 

25.90(a) 



(a)  At  this  time  sprouts  were  starting  upon  both  lots. 


It  will  be  seen  that  corn  absorbs  water  quite  rapidly  at  first, 
then  slowly  until  the  water  content  is  sufficient  to  cause  germi- 
nation, which  starts  at  a little  below  30  per  cent. 

It  is  doubtful,  in  view  of  this,  if  the  percentage  increase  of 
water  in  the  whole  seed  can  be  attributed  to  absorption  from 
air,  or  if  the  great  increase  of  water  in  the  embryo  is  due  to 
a direct  transfer  from  the  starchy  portion.  The  most  satisfac- 
tory explanation  both  for  the  increased  per  cent  of  water  in  the 
whole  seed  and  for  its  uneven  distribution  as  germination  pro- 
ceed?, is  the  production  of  metabolic  water  by  oxidation  of  or- 
ganic matter,  incident  to  respiration  of  the  embryo.  This  view 
is  sustained  by  a considerable  loss  of  organic  matter,  especially 
in  the  embryo. 

It  is  well  known  that  the  respiration  of  a germinating  seed 
is  chiefly  due  to  the  vital  activity  of  protoplasm,  within  the  em- 
bryo. In  consequence  of  this,  the  easily  oxidized  carbohydrates 


Role  op  Metabolic  Water  in  Vital  Phenomena  117 


contained  in  the  embryo  are  the  first  to  disappear,  being  con- 
verted into  carbon  dioxide  and  water  before  other  parts  of  the 
seed  are  attacked.  The  carbon  dioxide  produced  soon  escapes 
into  the  air,  while  the  water,  for  the  most  part,  remains  in  the 
tissues  of  the  embryo  where  it  is  formed. 

This  local  substitution  of  water  for  organic  matter  reduces 
materially  the  concentration  of  the  cell  fluids  thereby  disturbing 
the  osmotic  equilibrium  between  the  embryo  and  other  parts 
of  the  seed.  In  consequence  of  this,  there  is  a diffusion  of  or- 
ganic matter  into  the  live  cells  of  the  embryo,  and  to  a con- 
siderable extent  also  of  water  in  the  opposite  direction,  but 
since  organic  matter  within  the  embryo  is  continually  destroyed 
by  oxidation,  or  removed  from  solution  by  being  converted  in- 
to insoluble  nutrients  or  permanent  tissues  of  the  plant,  the 
concentration  of  the  fluids  of  a growing  cell  remains  constantly 
less  than  that  of  the  fluids  in  other  parts  of  a seed.  Organic 
nutrients  must  therefore  continually  move  towards  the  embryo 
so  long  as  the  protoplasm  is  active,  and  water,  metabolic  water, 
arising  from  the  oxidation  of  these  nutrients,  must  also  accumul- 
ate at  this  point.  It  is  chiefly  this  local  production  of  water 
within  the  growing  cells  that  induces  turgidity  and  pressure  up- 
on which  the  growth  of  new  cells  depends,  rather  than  upon  a 
movement  of  absorbed  water,  by  diffusion,  towmrds  the  growing 
points. 

If  a germinated  kernel  of  com,  upon  which  sprouts  have  de- 
veloped to  a length  of  from  one  to  two  inches,  be  exposed  to 
diffused  light  in  moderately  dry  air,  that  part  of  the  sprout 
next  to  the  seed  soon  withers  and  apparently  dies  while  the 
sprout  remains  succulent  and  continues  to  grow  at  the  tip  for 
several  days.  It  is  improbable  that  there  is  a transfer  of  either 
water  or  organic  nutrients,  through  the  dead  tissue,  from  the 
seed  to  the  living  part  of  the  sprout.  The  more  active  cells 
towards  the  tip  of  the  sprout  derive  their  nutriment  partly 
from  the  older  cells  near  the  base  which  then  die  of  starvation, 
and  partly  from  slow  photosynthesis.  The  water  already  in 
the  stem,  together  with  the  metabolic  water  derived  from  the 
oxidation  of  these  nutrients,  is  sufficient  to  maintain  the  vital 
functions,  in  a portion  of  the  cells,  for  a considerable  time,  in 
spite  of  a constant  loss  of  water  incurred  through  evaporation 
and  respiration,  and  of  water  used  in  photosynthesis  during 
the  day.  The  dead  section  becomes  longer  and  longer  as  the 


118  Wisconsin  Research  Bulletin  No.  22 

time  of  exposure  is  extended  until  finally  the  whole  sprout  is 
dry  and  dead,  but  so  long  as  the  tip  remains  alive,  its  water 
content  is  maintained  at  about  80  per  cent.  The  high  per 
cent  of  water  maintained  in  the  living  cells  is  unquestionably 
due,  in  large  measure,  to  metabolic  water  produced  by  oxida- 
tion of  organic  matter  that  is  transferred  by  osmosis  at  first 
from  the  seed  and  later  from  the  lower  portions  of  the  sprout. 
The  gradual  increase  in  the  percentage  of  dry  matter  in  the 
starchy  portion  of  a seed,  coincident  with  an  increase  of  water 
in  the  embryo  (See  Tables  IX  and  IXA),  as  germination  pro- 
ceeds, might  be  explained  by  transfer  of  water  from  the  sur- 
rounding tissues  into  the  embryo,  or  by  a movement  of  organic 
matter  in  the  opposite  direction,  but  a consideration  of  the 
physical  principles  involved,  and  of  the  chemical  reactions, 
known  to  occur  during  germination,  render  this  highly  im- 
probable. There  is  every  reason  to  believe  that  the  prevailing 
movement  of  free  water,  in  ar  germinating  seed,  up  to  the  time 
that  photosynthesis  begins,  is  away  from,  rather  than  towards, 
the  embryo;  and  that  the  excess  of  water  in  the  embryo  is  due 
to  local  oxidation  of  organic  matter  brought  into  it  by  osmosis, 
and  not  to  direct  diffusion  of  water  from  surrounding  tissues. 

The  percentage  increase  of  organic  matter  in  the  starchy  por- 
tion of  a seed,  at  this  time,  may  be  explained  by  the  hydrolytic 
reactions  that  always  occur  in  this  part  of  a germinating  seed, 
whereby  water  unites  with  starch  and  other  insoluble  carbohy- 
drates, as  well  as  with  insoluble  proteins,  to  form  soluble  and 
diffusible  products.  These  reactions  necessarily  precede  dif- 
fusion and  in  the  early  stages  of  germination  must  increase  the 
per  cent  of  organic  matter  in-  these  tissues,  especially  if  direct 
absorption  of  water  from  contact  with  a wet  surface  is  pre- 
vented, as  was  the  case  in  these  trials. 

This  condition  must  prevail  so  long  as  hydrolysis  of  the  re- 
serve nutrients  of  a seed  proceeds  at  a more  rapid  rate  than 
diffusion  of  organic  products  to  the  embryo  occurs,  since  oxida- 
tion of  organic  matter  does  not  take  place  in  the  starchy  por- 
tion of  a seed!  In  consequence  of  this,  there  is,  in  the  early 
stages  of  germination,  an  increase  in  the  absolute  as  well  as  in 
the  percentage  amount  of  organic  matter  in  the -starchy  por- 
tion of  a seed. 

A further  result  of  these  reactions  is  a permanent  difference 
between  the  concentration  of  soluble  nutrients  in  the  fluids  of 


Role  of  Metabolic  Water  in  Vital  Phenomena  119 

the  embryo  and  in  the  fluids  of  other  parts  of  the  seed,  the  con- 
centration always  being  less  in  the  embryo.  This  difference 
necessarily  causes  a movement  of  these  nutrients  by  osmosis 
towards  the  embryo,  where  they  are  either  oxidized,  or  deposit- 
ed as  new  tissue  containing  a lower  per  cent  of  water.  No  mat- 
ter which  direction  the  reaction  takes,  the  result  is  an  accumu- 
lation of  water  in  the  growing  cells. 

The  metabolic  water  resulting  from  these  reactions  is  con- 
siderable and,  in  spite  of  a transfer  of  water  to  other  tissues, 
and  of  evaporation  into  the  air,  the  sprouts  growing  upon  the 
soaked  seed  contain  between  80  and  90  per  cent  of  water.  This 
large  excess  of  water  in  the  growing  sprouts,  is  not  due  to 
capillarity,  since  with  no  external  water  supply  the  capillary 
forces  must  be  in  equilibrium  and  the  tendency  for  movement 
be  equal  in  both  directions;  nor  can  it  be  caused  by  the  more 
hygroscopic  nature  of  the  substances  composing  the  cells  of 
the  sprouts,  or  their  contents,  for  if  respiration  be  suspended 
by  exclusion  of  oxygen,  by  the  action  of  poisons,  or  of  anaesthe- 
tics, by  heating  or  by  freezing,  the  growing  sprouts  by  reason  of 
the  thinner  cell  walls  dry  much  more  quickly  upon  exposure 
than  do  the  original  tissues  of  the  seed.  It  appears  therefore, 
that  the  accumulation  of  water  in  the  active  cells  of  a sprouted 
seed  is  independent  of  either  the  physical  structure  or  the  chem- 
ical nature  of  the  tissues,  but  is  most  intimately  associated  with 
direct  respiration,  a vital  process  manifested  by  the  absorption 
of  oxygen  and  the  evolution  of  carbon  dioxide. 

Since  respiration  is  a function  peculiar  to  living  protoplasm, 
the  carbon  dioxide  evolved  must  be  derived  from  the  oxidation 
of  organic  nutrients  that  are  within  the  respiring  cells,  the  only 
place  where  active  protoplasm  is  found.  In  addition  to  the  car- 
bon dioxide  evolved,  there  is  produced,  at  the  same  time,  within 
these  cells  a considerable  quantity  of  water,  the  amount  depend- 
ing upon  the  nature  of  the  substance  oxidized  and  the  complete- 
ness of  the  reaction.  In  a.  germinating  kernel  of  corn,  the  oxi- 
dized substance  consists  chiefly  of  dextrose  which  has  been  de- 
rived from  the  stored  starch  of  the  seed  by  the  action  of  diastase. 
The  complete  oxidation  of  dextrose  results  in  the  production  of 
water  amounting  to  60  per  cent  of  the  weight  and  to  nearly  the 
same  volume  as  the  crystallized  dextrose.  But  the  amount  of 
water  resulting  from  the  complete  oxidation  of  sufficient  dex- 


120 


Wisconsin  Research  Bulletin  No.  22 


trose . to  account  for  all  of  the  carbon  dioxide  evolved,  by  no 
means  represents  the  total  water  produced  within  these  respir- 
ing cells,  since  this  reaction,  in  a growing  cell,  is  always  associa- 
ted with  other  reactions  that  not  only  remove  dextrose  from 
solution  but  at  the  same  time  liberate  water.  Thus  cellulose  is 
always  produced  within  these  cells  and  frequently  starch  is  also 
formed,  both  of  which  are  derived  from  dextrose.  If  only  the 
initial  substance  and  the  final  products  axe  considered,  the  re- 
action in  both  of  these  cases  may  be  expressed  as  follows : 

C6H1206  (dextrose)  = C6H10O5  (cellulose  or  starch)  + H20.  ' 
Besides  starch  and  cellulose,  cane  sugar  is  always  found  in 
varying  amounts  in  the  growing  sprouts  of  corn,  and  this  also 
must  have  originated  from  dextrose.  In  this  case,  only  half 
as  much  water  is  liberated  as  when  cellulose  or  starch  are  the 
products.  The  final  result,  when  cane  sugar  is  derived  from 
dextrose,  may  be  represented  as  follows : 

2 'C6H1206  (dextrose)  = C12H22On  (cane  sugar)  + H20. 
Reactions  analogous  to  these  in  which  no  carbon  dioxide  is 
liberated  are  always  taking  place  not  only  in  the  sprouts  of  a 
germinating  seed  but  in  all  tissues  of  plants,  in  all  stages  of 
development. 

There  is  no  way  of  determining  how  many  times  the  same 
carbon  nucleus  may  function  as  a carrier  of  water  in  these 
ways.  It  is  evident,  however,  that  metabolic  water  liberated 
within  the  respiring  cells  by  the  oxidation  and  dehydration  of 
organic  nutrients  brought  to  them  by  osmosis,  is  a most  impor- 
tant and  probably  the  chief  factor  concerned  in  the  movement  of 
water  from  the  leaves  where  organic  nutrients  are  primarily 
formed,  to  the  growing  centers.  It  is  amply  sufficient,  so  long 
as  proper  nutrients  are  supplied,  to  not  only  maintain  the  high 
water  content  of  these  tissues,  but  also  to  induce  sap  pressure 
and  turgidity  in  spite  of  considerable  losses  by  evaporation  and 
by  diffusion  of  water  to  other  portions  of  a plant. 

The  conditions  prevailing  in  sprouting  seed,  which  receives 
no  wTater  from  an  external  source,  are  all  opposed  to  a direct 
transfer  of  liquid  water  from  the  dead  portion  to  the  growing 
sprouts. 

In  a capillary  system  the  liquid  comprising  the  sap  must  be 
in  equilibrium ; the  concentration  of  the  nutrient  solution  is  far 
greater  in  the  older  tissues  where  the  reserve  nutrients  are 


Role  of  Metabolic  Water  in  Vital  Phenomena  121 

made  available  than  in  tlie  growing  sprouts  where  oxidation 
and  deposition  continually  occur;  and  the  sap  pressure  in  the 
active  cells  is  always  high  compared  to  that  in  the  dead  tissues 
of  the  seed.  Under  these  conditions  water  must  naturally  flow 
away  from  rather  than  towards  the  respiring  cells,  if  the  move- 
ment depends  solely  upon  physical  forces  and  the  water  is 
transferred  in  the  liquid  state  only. 

On  the  other  hand,  every  condition  favors  a movement  of 
soluble  organic  matter  in  this  direction  by  osmosis  and  all  of 
the  observed  chemical  changes  in  the  seed  and  sprouts  are  in 
accord  with  it.  In  the  respiring  cells  these  organic  nutrients 
are  oxidized  or  dehydrated,  sufficient  water  being  liberated  to 
supply  all  needs,  so  long  as  nutrients  are  available  and  evapora- 
tion is  not  excessive. 

Nutrients  And  Germination 

Among  the  conditions  essential  to  germination  is  a sufficient 
supply  of  suitable  organic  nutrients,  in  a soluble  form,  within  a 
seed,  to  maintain  respiration  of  the  active  cells  of  the  embryo. 
As  a rule,  air  dry,  viable  seeds  contain  a considerable  excess  of 
such  nutrients  which  are  mostly  insoluble  and  unavailable,  un- 
til water  is  provided  for  their  solution  and  distribution.  When 
a seed  is  placed  in  contact  with  about  half  its  weight  of  water, 
or  is  surrounded  by  moist  soil,  it  absorbs  enough  water  for 
these  purposes  and  respiration  is  greatly  stimulated  by  the  in- 
creased food  supplied  to  the  embryo.  If  however  a seed  be 
immersed  in  a larger  quantity  than  it  can  absorb,  some  of  its 
soluble  nutrients  are  lost  by  diffusion  into  the  excess  of  water, 
and  when  the  amount  of  water  is  very  large,  compared  to  the 
weight  of  the  seed,  the  soluble  nutrients  remaining  in  the  seed 
may  be  too  small  to  support  normal  respiration  and  the  seed 
will  either  send  out  feeble  sprouts,  or  may  not  germinate  at 
all-  If  the  water  surrounding  a seed  be  constantly  renewed,  as 
may  happen  during  a prolonged  rain,  soon  ofter  planting,  all 
of  the  soluble  nutrients  will  be  washed  out  and  the  seed  will 
•ail  to  germinate;  in  this  case,  germination  fails  because  of  ° 
lack  of  sufficient  soluble  nutrients  in  the  embryo  to  support  res- 
piration. The  following  experiments  confirm  these  statements  : 

A sample  of  corn  from  a lot  that  germinated  perfectly,  in 
contact  with  moist  filters,  was  placed  in  a flask  with  about 


122 


Wisconsin  Research  Bulletin  No.  22 


twenty  times  its  weight  of  boiled  water;  the  flask  was  fre- 
quently agitated  to  insure  distribution  of  extraetive  matter  and 
after  a few  hours  the  water  was  poured  off  and  replaced  by  a 
fresh  portion.  This  renewal  of  water  was  repeated  six  times  in 
forty-eight  hours.  The  first  extract  reduced  Fehlings  solution 
to  a considerable  degree  and  each  subsequent  extract  less,  the 
last  having  very  slight  effect,  indicating  that  nearly  all  soluble 
carbohydrates  had  been  removed  from  the  corn.  At  the  end  of 
forty-eight  hours,  some  of  these  soaked  kernels  were  placed  be- 
tween moist  filters  for  germination;  after  twenty-four  hours, 
ncne  of  these  kernels  were  germinated,  but  after  forty-eight 
hours  80  per  cent  were  germinated  with  weak  abnormal  sprouts, 
and  no  further  germination  occurred.  Seeds  from  the  same  lot 
that  were  soaked  for  seventy-two  hours  in  just  sufficient  water 
to  cover  them,  all  germinated  with  strong  healthy  sprouts  after 
only  twenty-four  hours  exposure  to  air  in  moist  filters. 

Another  portion  of  the  same  corn  was  soaked  twenty-four 
hours  longer,  seventy-two  hours  in  all,  and  then  tested  for 
germination  in  filters  moistened  with  pure  water,  in  filters  moist- 
ened with  a 5 per  cent  solution  of  dextrose,  and  in  a similar  solu- 
tion of  dextrose  to  which  a little  active  diastase  was  added.  The 
results  are  shown  in  Table  XI. 


TABLE  XI.  GERMINATION  OF  CORN  SOAKED  IN  BOILED  WATER 

The  sprouts  upon  the  seeds  that  were  germinated  with  water  only  were  far  weaker 
than  those  that  received  dextrose  in  addition. 


Hours  soaked 

Hours  of 
germination 
test 

Per  cent  germination 

In  water 

In  dextrose 

In  dextrose 
and  diastase 

0 

48 

24 

48 

30 

48 

72 

100 

0 

80 

40 

60 

60 

48 

48 

72 

60 

100 

70 

100 

72 

72 

Metabolic  Water  in  Mature  Plants 

Reactions  by  which  the  elements  of  water  are  tied  up  in  or- 
ganic combination  to  be  liberated  again  in  the  liquid  state,  at 
the  points  where  and  when  needed,  are  not  confined  to  germi- 
nating seeds  but  occur  in  all  stages  of  growth,  in  every  variety 
of  plants  and  provide  an  efficient  means  for  the  storage  of  water 


Role  of  Metabolic  Water  in  Vital  Phenomena  123 


in  a non-volatile  but  immediately  available  form;  they  are  the 
chief  factors  in  the  movement  of  water  and  of  organic  nutrients 
from  one  part  of  a plant  to  another;  they  induce  turgidity  and 
sap  pressure  in  the  growing  shoots,  where  respiration  is  active, 
even  after  all  connections  with  an  external  water  supply  are 
broken,  and  thus  they  enable  a plant  to  withstand  long  periods 
of  drought,  or  other  adverse  conditions,  without  permanent  in- 
jury. These  are  all  most  important  functions  upon  which  the 
life  and  growth  of  plants  depend  and  it  is  questionable  if  vital 
processes  would  be  possible  in  multicellular  plants  without 
some  such  provision  for  the  conservation  and  transfer  of  water 
from  cell  to  cell. 

The  reactions  involved  in  these  transformations  are  all  ex- 
tremely complex  and  the  intermediate  stages  obscure.  In  a 
plant  they  occur  with  equal  facility  in  both  directions 
hydrolysis  and  dehydration  taking  place  even  in  the  same 
cell  at  different  times  under  slight  changes  in  conditions  which 
at  present  are  not  well  defined.  A few  hydrolytic  reactions  of 
a similar  nature  to  those  that  occur  in  plants,  such  as  the  con- 
version of  starch  into  dextrose,  may  be  brought  about  in  the 
laboratory  by  chemical  means;  but  up  to  the  present  time  very 
few  of  the  well  recognized  carbohydrates,  or  fats,  and  none  of 
the  proteins  have  been  produced  artificially,  by  dehydration  or 
other  treatment  of  a more  highly  hydrated  member  of  the  group 
to  which  it  belongs. 

Composition  of  Plant  Tissues 

The  dry  organic  tissues  of  plants  consist  chiefly  of  carbohy- 
drates, fats  and  proteins,  but  as  fats  are  primarily  derived  from 
carbohydrates,  and  in  the  metabolism  of  plants  are  probably 
converted  into  carbohydrates  and  since  there  appears  to  be  no 
destructive  metabolism  of  proteins,  it  is  sufficient  for  the  purpose 
of  this  paper  to  consider  only  the  relations  of  the  carbohydrate 
group. 

Carbohydrates  All  carbohydrates  consist  of  carbon  combin- 
ed with  oxygen  and  hydrogen,  the  amounts  of  oxygen  and  hy- 
drogen having  the  same  relation  to  each  other  as  in  water. 
The  first  well  defined  and  stable  carbohydrate  to  appear  in 
plants  is  starch,  which  is  formed  in  the  chlorophyl  bearing  cells 
of  leaves  by  the  action  of  light  upon  a solution  of  carbon  dioxide 


124 


Wisconsin  Research  Bulletin  No.  22 


in  water.  If  light  is  excluded,  or  in  its  presence  if  carbon 
dioxide  is  withheld,  the  starch  previously  formed  in  a healthy 
leaf  soon  disappears,  being  converted  by  specific  enzymes,  into 
dextrose  and  other-  soluble  and  diffusible  carbohydrates  of  a 
higher  degree  of  hydration  than  starch,  which  are  distributed 
throughout  the  plant  by  osmosis  and  serve  as  nutrients  for  the 
active  cells.  It  seems  likely  that  this  inversion  of  starch  occurs 
continually , even  in  sunlight  but  that  under  these  conditions, 
its  effect  is  masked  by  the  constant  production  of  starch  from 
carbon  dioxide  and  water. 

In  the  course  of  these  transformations  a great  variety  of  car- 
bohydrates is  formed,  the  most  sharply  defined  of  which  are  cel- 
lulose, (C6H10O5)n,  starch,  (CrH1005)n,  cane  sugar,,  (C19H220„) 
maltose  (C12H22On) , dextrose  and  levulose,  (C6H1206)~ 

Aside  from  the  above  mentioned  carbohydrates,  there  are  a 
number  of  intermediate  and  closely  allied  bodies  such  as  dex- 
trine, the  various  gums,  the  pectous  substances,  the  pentozans 
etc.,  the  molecular  structures  of  which  are  not  well  understood, 
but  which  have  an  important  role  in  the  carbohydrate  metabo- 
lism of  plants.  No  doubt  in  plants,  some  of  these  are  always 
formed  in  every  transformation  of  one  of  the  principal  carbo- 
hydrates to  a higher  or  a lower  degree  of  hydration,  since  they 
appear  in  every  stage  of  plant  development. 

Cellulose  This  is  the  most  stable  of  these  carbohydrates  and 
when  once  deposited,  in  a healthy  plant,  remains  permanently 
in  the  place  where  it  was  formed,  constituting  an  organic  frame- 
work which  supports  the  growing  cells  and  determines  the 
general  form  of  the  plant.  It  appears  to  be  produced,  in  the 
plant,  by  a partial  dehydration  of  dextrose,  and  other  soluble 
carbohydrates  derived  from;  starch,  a reaction  that  is  always 
associated  with  respiration  of  the  cells.  It  is  not  acted  upon 
by  any  of  the  enzymes  of  healthy  plants.  It  is  possible,  by 
the  action  of  proper  reagents,  to  hydrolyze  cellulose  and  produce 
some  of  the  more  highly  hydrated  members  of  the  carbohydrate 
group,  but  the  reaction  is  never  complete  and  the  theoretical 
amount  of  dextrose  is  never  obtained,  nor  is  starch  or  cane 
sugar  formed  among  the  products  of  the  reaction.  It  is  insolu- 
ble in  water  or  in  the  sap  of  plants. 

Starch  The  same  empirical  formula  is  given  to  starch  as 
to  cellulose.  Like  cellulose  it  is  a dyhydration  product  of  solu- 


Kole  of  Metabolic  Water  in  Vital  Phenomena  125 

ble  carbohydrates.  It  is  insoluble,  either  in  pure  water  or  m 
the  sap  of  plants,  and  is  the  form  in  which  reserve  carbohydrate 
nutrients  are  most  frequently  deposited  in  the  plant  tissue. 
It  is  readily  converted  by  diastase  into  the  theoretical  amount 
of  dextrose,  the  reaction  taking  place  with  equal  facility,  with- 
in or  without  the  plant.  The  same  change  may  be  brought 
about  in  the  laboratory  by  the  action  of  suitable  reagents  and 
is  therefore  not  dependent  upon  vital  processes.  The  reverse 
reaction  has  never  been  accomplished  outside  the  plant.  It  is 
also  possible  that,  in  a plant,  cane  sugar  may  be  formed  di- 
rectly from  starch. 

C (me  Sugar  This,  carbohydrate  stands  intermediate  be- 
tween starch  and  dextrose  in  the  amount  of  hydrogen  and  oxy- 
gen, (the  elements  of  water),  that  it  contains.  It  is  found  in 
every  part  of  most  chloropliyl  bearing  plants,  in  every  stage 
of  their  development,  from  the  sprouting  seed  to  the  mature 
fruit.  This  indicates  that  it  is  a direct  product  of  respiration, 
being  formed,  in  some  unexplained  manner,  in  all  active 
cells,  from  the  dextrose  or  maltose  that  results  from  the  hydro- 
lysis of  starch.  It  may  serve  as  a reserve  carbohydrate  nutri- 
ent in  an  analogous  manner  to  starch,  and  like  starch  cannot 
he  directly  utilized  by  cells  as  a source  of  energy  for  maintain- 
ing vital  activity  until  it  has  been  hydrolized  and  converted  into 
invert  sugar,  maltose,  or  some  other  easily  oxidized  carbohy- 
drate. This  change  may  be  brought  about  in  any  tissues  of  a 
plant  but  is  usually  effected  in  the  leaves  where  conditions  ap- 
pear to  be  the  most  favorable,  especially  when  exposed  to 
light. 

This  function  of  leaves,  by  which  cane  sugar  is  hydrolized 
during  photosynthesis  is  shown  by  the  low  per  cent  of  cane 
sugar  found  in  leaves,  compared  to  that  in  other  tissues,  as 
well  as  by  its  high  per  cent  in  the  sap  of  sugar-producing 
plants,  such  as  sugar  cane,  Indian  corn  and  sugar  beets,  as 
maturity  approaches  and  the  leaves  become  less  active.  Similar 
conclusions  may  be  drawn  from  the  observations  of  Keitt 7 upon 
the  sugar  content  of  sweet  potatoes,  who  found  more  cane  sugar 
in  potatoes  harvested  after  a wet  and  cloudy  period  than  after 
a period  of  fair  weather.  The  cane  sugar  that  accumulates  at 
maturity,  and  during  resting  periods  when  photosynthesis  is 


• Bui.  156  S.  C.  Exp.  Sta. 


126 


Wisconsin  Research  Bulletin  No.  22 


suspended,  usually  serves  as  a reserve  nutrient  and  disappears 
when  leaves  again  appear.  Thus  the  cane  sugar  stored  in  the 
beet  root,  at  the  end  of  the  first  seasons  growth,  rapidly  disap- 
pears when  the  leaves  form  in  the  following  spring.  The  slow 
respiration  that  occurs  during  winter,  in  the  cells  of  most  de- 
ciduous trees,  converts  a large  part  of  the  stored  starch  into 
cane  sugar  which  is  inverted  and  made  available  as  soon  as 
leaves  expand  in  spring. 

Cane  sugar  diffuses,  as  it  is  formed!,  from  the  active  cells 
into  the  vessels  which  transfer  water  from  the  roots  to  the  leaves 
and  for  the  most  part,  is  carried  by  this  current  of  water,  to- 
gether with  carbon  dioxide  and  other  products  of  respiration, 
to  the  leaves,  where  all  are  converted  by  photosynthesis,  into 
available  nutrients  and  returned  by  osmosis  to  the  active  cells. 
This  cycle  may  be  repeated  an  indefinite  number  of  times. 

In  this  way,  cane  sugar  and  similar  carbohydrates  serve  by 
means  of  alternating  hydrolytic  and  dehydrating  reactions  as 
most  important  agents  in  the  transfer  of  water  from  the  leaves 
to  all  of  the  growing  cells  of  a plant.  The  amount  of  water 
transferred  in  this  manner  is  unknown,  since  no  means  are 
available  for  determining  the  number  of  times  which  the  same 
carbon  atoms  function  in  this  role.  I believe  that  the  water 
abstracted  from  leaves,  in  this  way,  is  nearly  as  potent  a factor 
m causing  leaves  to  wilt  in  sunlight  as  is  direct  transpiration 
of  water  from  the  leaves. 

Maltose  The  same  empirical  formula  (Ci9H2^01:l),  repre- 
sents  maltose  and  also  cane  sugar;  it  is  formed,  with  dextrose, 
by  the  action  of  diastase  upon  starch;  it  is  soluble  in  water 
and  m the  sap  of  plants ; it  reduces  Fehlings  solution 
readily.  Maltose  is  quite  widely  distributed  in  the  vegeta- 
ble kingdom  and  undoubtedly  serves  as  a direct  nutrient  for 
the  support  of  respiration;  in  this  respect  it  differs  radically 
from  its  isomer,  cane  sugar,  since  this  is  only  available  after 
inversion  by  ferments  or  acids  to  dextrose  and  levulose. 

Dextrose  and  Levulose  These  carbohydrates  are  similar  in 
their  chemical  relations  and  represent  the  highest  state  of  hy- 
dration of  any  that  occurs  in  plants.  Both  are  soluble  in  water, 
are  readily  diffusible,  and  reduce  Fehlings  solution.  It  is  in 
these  forms  that  carbohydrate  nutrients  are  most  frequently 
transferred  from  place  to  .place,  in  a plant,  although  cane  sugar 


Role  of  Metabolic  Water  in  Vital  Phenomena  127 


serves  this  purpose  to  a limited  extent.  They  also  serve  as  im- 
portant agents  for  the  transfer  of  water  to  the  growing  cells. 
In  a living  plant;  all  other  members  of  the  carbohydrate  group 
may  be  derived  from  them  by  dehydration  and,  conversely,  all 
other  carbohydrates  except  possibly  cellulose  may  be  converted 
into  these  by  hydrolysis. 

The  importance  of  these  properties  of  carbohydrates  by  which 
they  may  be  changed  from  one  degree  of  hydration  to  another, 
alternating  between  starch  on  the  one  hand  and  dextrose  or 
levulose  on  the  other,  is  illustrated  by  the  transformations,  al- 
ready mentioned,  that  precede  and  accompany  the  germination 
of  seeds.  There  is  first,  the  absorption  of  water  from  an  exter- 
nal source,  in  sufficient  quantity  to  permit  the  action  of  enzymes 
upon  the  stored  nutrients  and  for  the  transfer  of  soluble  pro- 
ducts by  osmosis  to  the  active  cells  of  the  embryo,  where  they 
stimulate  respiration  and  are  in  part  oxidized  to  carbon  dioxide 
and  water,  and  in  part  dehydrated  to  form  lower  members  of 
the  carbohydrate  groups,  containing  less  of  the  elements  of  wafer 
than  before.  In  either  case  the  organic  nutrients  that  are 
brought  to  the  respiring  cells  are  partly  replaced  by  water,  so 
that  the  solution  of  nutrients  within  these  cells  is  constantly 
maintained  at  a lower  concentration  than  in  the  surrounding 
tissues.  In  this  way  conditions  favorable  to  a continuous 
movement  of  nutrients  towards  the  depleted  centers,  where 
growth  occurs,  are  maintained,  so  long  as  the  cells  remain  alive 
and  a store  of  suitable  nutrients  is  available. 

Bulbs  and  Tubers 

All  bulbs  and  tubebs  undergo  transformations  similar  to  the 
ones  which  seeds  undergo ; they  constantly  respire,  absorbing 
oxygen,  evolving  C02,  and  producing  water  within  the  tissues. 
Respiration  occurs  chiefly  at  the  growing  centers,  as  in  the  cen- 
tral bud  of  an  onion,  or  in  the  eyes  of  a potato.  The  enzymes, 
located  at  or  near  these  centers  act  upon  the  stored  food  pro- 
ducts, in  the  bulb  or  tuber,  converting  them  into  soluble  and 
diffusible  substances,  which  in  turn  are  oxidized  in  the  develop- 
ing cells,  thereby  raising  their  water  content  and  increasing 
their  turgidity  to  a point  where  growth  is  induced. 

If  a bulb  like  an  onion  be  immersed  in  water  which  has  been 
boiled  to  expel  air?  it  does  not  sprout,  nor  does  it  appear  to 


128 


Wisconsin  Research  Bulletin  No.  22 


absorb  water.  If,  however,  the  bulb  be  exposed  to  warm,  moist 
air,  it  soon  sprouts  and  grows  to  a considerable  extent  upon  the 
stored  nutrients  which  the  bulb  contains.  In  this  case  the 
sprouts  contain  a higher  proportion  of  water  than  was  origin- 
ally present  in  the  bulb.  The  excess  of  water  in  the  sprouts 
is  chiefly  due  to  metabolic  water  formed  from  the  organic  nutri- 
ents by  respiration.  The  fleshy  tissues  of  a bulb  serve  not  only 
to  supply  nutrients  to  the  developing  bud,  but  also  to  protect  it 
from  a too  abundant  supply  of  oxygen,  until  conditions  are 
favorable  for  growth. 

A similar  provision  is  found  in  fruits,  in  which  the  pulpy 
material  protects  the  seeds  from  free  access  of  oxygen  and  re- 
duces respiration  of  the  seeds  to  a point  where  germination  can- 
not occur  until  the  easily  oxidized  constituents  which  surround 
them  are  destroyed.  In  most  cases,  this  maintains  the  seed  in  a 
nearly  dormant  condition  until  the  following  spring,  when  germi- 
nation occurs  quickly  and  the  plant  has  a whole  season  to  develop 
and  mature  its  woody  tissues  and  buds  before  winter.  Were  it  not 
for  this  protection,  many  seeds  would  germinate  in  late  summer 
and  the  immature  growth  would  be  killed  by  freezing.  The  influ- 
ence of  the  pulpy  substance  of  fruit,  in  delaying  germination 
of  seeds  is  seen  in  melons  and  similar  fruits  the  seeds  of  which 
do  not  germinate  so  long  as  the  fruit  is  unbroken,  although 
temperature  and  moisture  conditions  may  be  ideal  for  growth, 
but  when  seeds  are  removed  from  the  fruit  and  placed  in  moist 
soil,  or  between  wet  filters,  in  contact  with  air,  they  germinate 
quickly.  Even  when  the  fruit  is  broken  so  that  air  has  free  ac- 
cess to  the  seeds,  some  seeds  germinate  in  a short  time,  if  molds 
are  suppressed,  showing  that  the  one  condition,  essential  to 
growth,  that  is  absent,  is  a supply  of  oxygen.  Germination  of 
fresh  seeds  from  pulpy  fruits  like  melons  is  facilitated  by  thor- 
oughly washing  the  seeds  to  remove  the  adhering  slimy  material ; 
unless  this  is  done  molds  quickly  appear  in  abundance  and  pre- 
vent germination.  The  best  results  have  been  obtained  wdien 
the  washed  seeds  were  placed  between  filter  papers  that  were 
moistened  with  a 3 per  cent  solution  of  hydrogen  peroxide, 
which  supplies  oxygen  in  abundance  and  keeps  molds  from 
gaining  the  ascendency,  until  after  the  sprouts  start.  Treated 
in  this  way  the  fresh  melon  seeds  germinate  nearly  as  quickly 
And  as  well  as  dried  seeds. 


Role  of  Metabolic  Water  in  Vital  Phenomena  129 


Viability  of  Immature  Seeds 

Seeds  seldom  germinate  while  they  are  attached  to  the  succu- 
lent living  tissues  of  the  parent  plant,  although  temperature 
and  moisture  conditions  are  usually  favorable  at  this  time.  The 
failure  to  grow  may  be  due  to  exclusion  of  free  oxygen  by  the 
seed  envelope,  and  it  may  be  that  organic  nutrients,  in  a form 
suitable  for  the  growing  sprout,  are  absent  at  this  time.  What- 
ever the  direct  cause  may  be,  the  adverse  conditions  disappear 
after  the  immature  seed  is  exposed  to  air  for  a period  that  de- 
pends upon  its  variety  and  maturity. 

Radish  seed,  taken  from  green  seed  pods,  all  failed  to  ger- 
minate, when  transferred  directly  from  the  pod  to  wet  filters, 
but  seed  from  the  same  lot,  after  being  kept  ten  days  in  warm 
dry  air,  all  germinated  within  forty-eight  hours,  under  the  same 
conditions. 

Corn  that  was  apparently  mature,  but  picked  while  the  husks 
were  still  green,  behaved  in  a similar  manner  to  the  radish  seed. 
Not  a single  kernel  sprouted  when  tested  immediately  after 
picking.  All  grew  after  a preliminary  ten  days7  exposure 
to  warm,  dry  air.  The  same  result  was  obtained  with  sweet 
corn,  picked  in  an  edible  condition  while  the  kernels  were  still 
soft  and  milky. 

In  the  case  of  corn,  this  change  in  viability  is  not  due  to  pre- 
liminary drying,  since  soft  kernels  from  the  same  ears,  when 
immersed  in  hydrogen  peroxide,  all  germinated  within  two 
weeks,  without  having  been  dried  at  any  time.  This  indicates 
that  direct  respiration  is  the  chief  factor  in  bringing  about  those 
changes  in  the  seed,  that  are  essential  to  germination.  Further 
confirmation  of  this  view  is  supplied  by  failure  of  such  seeds 
to  germinate  after  being  kept,  for  a similar  period,  in  carbon 
dioxide  or  in  boiled  water,  where  no  free  oxygen  was  available. 

Development  of  Hydrolytic  Ferments  in  Seeds 

Kernels  of  corn,  from  the  same  ears  described  above,  were 
tested  for  diastatic  ferments,  when  first  picked,  and  again  when 
germination  tests  were  afterwards  made.  For  this  purpose,  the 
crushed  kernels  were  digested  for  a few  hours  in  water  to  which 
a little  toluol  or  chloroform,  was  added  to  inhibit  fermentation ; 
to  the  clear  filtrate  from  this  mixture,  sufficient  starch,  in  solu- 
tion, was  added  to  give  a faint  but  distinct  blue  color  with  iodine ; 


130 


Wisconsin  Research  Bulletin  No.  22 


the  solution  was  kept  warm  and  tested  with  iodine,  at  frequent 
intervals.  A control  solution  in  water  only,  containing  the  same 
amount  of  starch,  was  exposed  and  tested  in  the  same  manner ; 
if  no  difference  in  the  intensity  of  the  reaction,  with  these  solu- 
tions, was  observed  after  two  hours,  it  was  assumed  that  diasta- 
tic  ferments  were  absent  from  the  seed.  In  all  tests  made  with 
corn,  after  it  had  been  picked  sufficiently  long  to  germinate  well, 
the  starch  reaction  disappeared  in  a short  time,  indicating  the 
presence  of  a starch  inverting  enzyme.  In  only  one  test  of  soft 
corn,  that  of  an  ear  with  the  husks  dry  when  picked,  was  any 
indication  of  a diastatic  ferment  found. 

A summary  of  a number  of  these  tests  is  given  in  Table  XII. 
The  water  content  of  samples  of  corn  tested  is  also  given  to  show 
the  relative  maturity  of  the  seed. 


TABLE  XII.  GERMINATION  OF  MATURE  AND  IMMATURE  SEEDS 

Influence  of  maturity  and  Exposure  to  air  upon  germination  and  upon 
the  presence  of  a diastatic  ferment  in  the  seed. 


I 

Per 

Percent  germination 

Variety  of  seeds 

Diastatic 

State  of  maturity 

cent 

water 

In  wet 

In  hydrogen 

ferment 

filters 

peroxide 

Yellow  dent  corn 

Ripe,  one  year  old.. 

8.50 

100  in 

100  in 

Present  in 

Yellow  dent  corn 

48  hours. 

48  hours. 
100  after 

abundance. 

Ripe  but  soft. .' 

40.62 

o 

14  days 
100  after 

Small  amount 

Y ellow  dent  corn 

Ripe  but  soft 

54.22 

o 

14  days. 

Doubtful 

Stowell’s  Evergreen 

sweet  corn 

In  edible  condition. 

74.71 

0 

None 

Radish 

From  green  pods. . . 

0 

Radish 

From  same  green 
pods  as  above  af- 

100 in 
48  hours, 

ter  10  days. 

After  being  exposed  to  warm  dry  air  for  ten  days,  seed  from 
each  of  the  ears  tested  as  shown  in  Table  XII  germinated  with- 
in forty-eight  hours  in  wet  filters,  and  also  when  immersed  in  a 
solution  of  hydrogen  peroxide. 

These  tests  indicate  that  the  presence  of  certain  specific  en- 
zymes is  essential  to  the  germination  of  seeds,  and  also  that  the 
production  of  such  enzymes  occurs  only  under  conditions  which 
admit  of  direct  respiration.  The  great  increase  in  respiration, 
as  well  as  in  the  production  of  such  enzymes  at  the  time  of  ger- 
mination gives  support  to  this  view. 


Role  of  Metabolic  Water  in  Vital  Phenomena  131 


The  course  of  metabolism  in  an  immature  seed  differs 
widely  from  that  in  a germinating  mature  seed.  In  the  former 
case,  soluble  nutrients  are  being  converted  into  insoluble  re- 
serve materials,  while  in  the  latter  case  the  reactions  are  re- 
versed, since  the  sprouting  embryo  must  receive  its  nutrients  in 
a highly  hydrated  and  soluble  form.  It  is  not  strange,  there- 
fore, that  hydrolytic  enzymes  are  wholly  absent  from  immature 
seeds,  so  long  as  a surplus  of  suitable  organic  nutrients  is  sup- 
plied through  the  circulatory  system  of  the  parent  plant,  and 
that  they  are  formed  only  after  direct  and  independent  respira- 
tion is  established. 

Unbroken  seeds  never  germinate  in  the  digestive  tract  of  an 
animal,  because  free  oxygen  to  support  direct  respiration  is  not 
available.  In  this  case,  temperature  and  moisture  conditions 
are  ideal  for  growth  and  when  voided,  in  the  excrement,  and 
exposed  to  air,  such  seeds  in  general  germinate  quickly. 

The  absorption  of  water  by  seeds,  the  conversion  of  starch 
into  dextrose  by  diastase,  the  distribution  of  dextrose  by  osmosis 
and  diffusion  throughout  the  water  of  the  seed,  and  all  other 
phenomena  that  precede  germination  take  place  with  equal  facil- 
ity in  dead  and  living  seeds.  In  fact  nearly  all  parts  of  a viable 
kernel  of  corn,  or  other  seed,  are  dead  and  may  be  removed  from 
the  embryo  without  affecting  the  development  of  new  tissue, 
provided  proper  nutrients  are  supplied  from  other  sources. 

The  very  slow  action  of  diastase  in  converting  starch  into  dex- 
trose, under  conditions  prevailing  in  an  air  dried  seed,  and  its 
greatly  increased  activity  when  an  abundance  of  water  is  sup- 
plied are  shown  by  the  following  experiment. 

Five  grams  of  coarsely  pulverized  corn  meal  were  boiled  in 
100  c.  c.  of  water  to  destroy  the  activity  of  the  diastase  con- 
tained in  the  meal.  To  another  similar  portion  of  the  same 
meal  was  added  100  c.  c.  of  cold  water.  A few  drops  of  toluol 
were  added  to  each  to  prevent  fermentation,  and  both  portions 
were  left  at  room  temperature  for  twenty-four  hours.  The  fil- 
trate from  the  cold  extract,  when  boiled  with  Fehlings  solution, 
gave  a copious  precipitate  of  cuprous  oxide,  while  scarcely  a 
trace  appeared  with  that  from  the  boiled  meal.  The  boiled  ex- 
tract contained  all  of  the  dextrose  that  had  accumulated  during 
several  months  in  the  air  dried  seed,  while  the  cold  extract  con- 
tained in  addition,  the  amount  of  dextrose  formed  in  only  twen- 


132 


Wisconsin  Research  Bulletin  No.  22 


ty  four  hours,  by  the  same  quantity  of  diastase,  in  the  presence 
of  sufficient  water  to  insure  its  maximum  activity. 

Molecular  Combination  of  Water  with  the  Constituents 

of  Seeds 

3 he  water  absorbed  by  seeds  before  germination  may  be  held 
by  purely  physical  forces,  such  as  are  manifested  in  capillarity 
and  adhesion,  or  it  may  be  in  molecular  combination  with  some 
of  the  organic  constituents  of  the  seed  as  appears  to  be  the  case 
m the  phenomena  of  imbibition.  The  removal  of  such  water  by 
exposure  to  a temperature  not  exceeding  100° C.  suggests  that 
physical  forces  only  are  involved,  but  the  persistence  with  which 
seeds  retain  water,  when  exposed  to  dry  air  at  ordinary  tem- 
peratures, indicates  that  at  least  a part  of  the  water  is  held  by 
feeble  molecular  combinations  analogous  to  that  in  crystals  con- 
taining water  of  crystallization. 


The  Nature  of  Imbibition 

Pile  term  imbibition  is  used  by  botanists  and  plant  phy- 
siologists to  designate  the  phenomena  associated  with  the  ab- 
sorption of  water  by  the  solid  material  of  plants.  The  most 
widely  accepted  hypothesis  concerning  these  phenomena  is  that 
first  advanced  by  Naegeli8  that  all  organized  tissues  of  plants, 
that  are  capable  of  imbibition,  consist  of  minute  molecular  aggre- 
gations designated  as  micellae,  between  which  water  enters,  forc- 
ing them  apart  thus  increasing  the  volume  of  the  tissues,  ’ when 
they  are  immersed  in  water.  The  entrance  of  water  between 
the  micellae  is  supposed  to  be  effected  by  some  other  force  than 
capillarity,  since  it  does  not  take  place  when  the  tissues  are  im- 
mersed in  such  liquids  as  absolute  alcohol  or  anhydrous  gly- 
cerin. 

fihis  argument,  which  is  advanced  to  show  the  inadequacy  of 
capillarity  to  explain  the  phenomena  of  imbibition,  appears  to 
me  to  be  equally  conclusive  against  the  existence  of  micellae. 
In  order  that  a liquid  may  be  absorbed  by  capillarity  it  is  es- 
sential that  the  absorbing  solid  and  the  absorbed  liquid  have 
some  affinity  for  each  other.  Most  liquids  will  rise  in  a capii- 


8 A clear  presentation  of  Naee:eli’s  views  and  tlie  evidence  in  support 
of  them  is  given  by  Sachs,  (Lectures  on  the  Physiology  of  Plants 
translated  by  H.  Marshall  Ward,  Lecture  XIII),  and  Pfeffer  (Physiol- 
ogy of  Plants,  translated  by  Alfred  J.  Ewart,  Chap.  HI). 


Role  of  Metabolic  Water  in  Vital  Phenomena 


133 


lary  tube  of  glass,  to  a point  above  the  level  outside,  but  mer- 
cury, which  has  no  molecular  attraction  for  glass,  only  enters  a 
capillary  tube  when  subjected  to  pressure.  The  same  principle 
is  involved  in  all  phenomena  of  this  nature,  and  all  liquids  which 
adhere  to  the  clean  surface  of  a solid  will  enter  a capillary  open- 
ing in  that  solid,  or  will  be  forced  between  the  limiting  surfaces 
of  such  solids  when  they  are  brought  as  nearly  in  contact  as 
possible  ; the  force  that  causes  the  liquid  to  enter  these  spaces 
is  an  inverse  function  of  the  distance  between  them.  The  fact 
that  plant  tissue  does  not  swell  when  immersed  in  absolute  al- 
cohol, or  anhydrous  glycerin,  both  of  which  adhere  to  the  clean 
dry  surface  of  bodies  composed  of  such  tissues,  is  conclusive/ 
evidence  of  the  inadequacy  of  Naegeli’s  hypothesis.  On  the 
other  hand,  all  of  the  phenomena  of  imbibition  point__directly 
to  a molecular  combination  'between  the  substance  composing  an 
organized  body~hRdWvater.  It  is  true  that,  in  most  cases,  the 
combination  is  feeble,  since  it  is  broken  up  by  a relatively  low 
temperature  without  changing  the  molecular  structure  of  either 
the  solid  tissue  or  the  water.  It  is,  however,  entirely  analogous 
to  the  behavior  of  many  substances,  both  organic  and  inorganic, 
which  crystallize  with  water  of  crystallization.  Such  substances 
exhibit  the  same  phenomena  in  combining,  viz.  a change  of  vol- 
ume, an  evolution  of  heat,  which  is  less  for  each  additional  in- 
crement of  water,  and  a tendency  towards  a uniform  distribution 
of  water  throughout  the  whole  mass,  when  saturated  and  un- 
saturated  portions  are  brought  in  contact. 

For  these  reasons,  it  seems  far  simpler  to  account  for  the 
phenomena  of  imbibition  by  a direct,  molecular  combination  of 
the  substances  composing  the  tissues  of  organized  bodies  and 
water,  than  by  assuming  the  existence  of  micellae,  the  structure 
and  form  of  which  cannot  be  demonstrated. 

Change  of  Temperature  wpien  Seeds  Absorb  Water 

Further  light  on  this  question  has  been  sought  by  determina- 
tions of  the  thermal  balance,  when  seeds  of  corn  are  moistened 
with  water  under  conditions  which  prevent  vital  activity.  If 
there  is  no  molecular  combination,  under  these  conditions,  there 
should  be  no  appreciable  disturbance  in  the  thermal  balance 
when  a seed  absorbs  water;  if,  on  the  other  hand,  the  absorbed 
water  is  chemically  combined  with  some  of  the  organic  constitu- 


134 


Wisconsin  Research  Bulletin  No.  22 

ents  of  the  seed,  it  should  be  indicated  by  a change  in  temper- 
ature. 

In  order  to  differentiate  the  reactions  involved  in  the  pre- 
liminary absorption  of  water  from  those  associated  with  vital 
processes,  an  antiseptic  was  added  to  the  water  with  which  the 
seeds  were  moistened.  This  not  only  suspended  all  vital  activity 
of  the  respiring  cells  of  the  seed,  but  also  prevented  the  growth 
of  molds  and  other  organisms  during  the  test.  On  the  other 
hand,  the  absorption  of  water  and  the  hydrolytic  .action  of  en- 
zymes upon  the  stored  nutrients  of  the  seed  were  not  interfered 
with. 

Two  varieties  of  corn,  a white  and  a yellow  dent,  both  of  the 
crop  of  1910,  were  selected  for  the  test. 

In  each  test  150  grams  of  corn  were  placed  in  a silvered  De- 
war  flask  of  500  c.  c.  capacity  with  200  c.  c.  of  water,  which  is 
sufficient  to  cover  the  seed,  and  5 c.  c.  of  toluol  to  prevent  fer- 
mentation and  suspend  respiration.  A sensitive  thermometer, 
graduated  to  0.1°C.  was  inserted  through  a perforated  rubber 
stopper  and  the  temperature  observed  at  frequent  intervals. 
During  the  test  the  flask  was  kept  in  a room  the  temperature  of 
which  was  approximately  the  same  as  the  contents  of  the  flask. 
Two  experiments  were  made  with  each  variety  of  corn.  In  each 
case  the  temperature  rose  quite  steadily  at  first,  reaching  a max- 
imum in  eight  to  ten  hours,  after  which  it  remained  constant  for 
the  next  30  hours  and  then  gradually  fell  off.  The  results  were 
concordant,  showing  a maximum  increase  in  temperature  of 
1.7°C.  for  the  yellow  corn,  and  of  2.6°C.  for  the  white  corn. 
Assuming  that  the  specific  heat  of  corn  is  0.3,  and  that  no  heat 
was  lost  by  radiation,  the  heat  change,  indicated  by  the  max- 
imum temperature  is  equivalent  to  3.25  calories  per  gram  for 
the  yellow,  and  to  4.25  calories  per  gram  for  the  white  variety. 
To  each  of  these  values  should  be  added  the  heat  absorbed  by 
the  apparatus  and  the  heat  dissipated  by  radiation,  during  the 
test,  the  amounts  of  which  are  unknown. 

At  the  end  of  the  test  the  water  content  of  the  yellow  corn 
was  46.94  per  cent,  and  of  the  white  corn  53.48  per  cent.  This 
difference  indicates  a higher  degree  of  hydration  of  the  white 
variety  and  explains,  in  a measure,  the  difference  in  the  amount 
of  heat  set  free. 

These  results  give  no  clue  to  the  particular  constituent  of  the 
seed  which  united  with  water  to  cause  the  rise  in  temperature, 


Role  of  Metabolic  Water  in  Vital  Phenomena 


135 


but  the  large  preponderance  of  starch  in  the  corn  grain  pointed 
to  this  as  the  most  probable  source. 

Confirmation  of  this  view  was  sought  by  comparing  the 
amounts  of  water  absorbed  by  starch  and  by  the  corn  grain, 
under  similar  conditions,  and  also  by  measuring  the  heat  evolved 
when  starch  was  mixed  with  water.  Previous  experiments  had 
shown  that  corn,  when  exposed  to  saturated  air,  absorbs  water 
rapidly  at  first,  then  more  slowly  until,  at  the  end  of  a week, 
it  contains  about  25  per  cent  of  water.  Changes  incident  to 
germination  begin  with  about  this  proportion  of  water  and  cause 
the  water  content  of  a live  seed  to  increase  more  rapidly  after 
this  time.  In  order  to  avoid  the  error  incident  to  vital  activity, 
even  when  the  time  was  extended  to  complete  saturation,  the 
absorption  of  water  by  kernels  of  corn  that  had  been  dried  at 
97° C.,  and  were  dead,  was  determined  for  comparison  with 
starch. 

About  5 grams  of  the  dried  corn  was  exposed  to  air  that  was 
saturated  with  moisture,  under  a bell  glass.  Complete  satura- 
tion of  the  air  was  secured  by  suspending  within  the  bell  glass, 
which  stood  in  a dish  of  water,  strips  of  filter  paper  with  the 

TABLE  XIII.  GAIN  OF  WEIGHT  BY  CORN  AND  STARCH  IN  MOIST  AIR 


Percentage  gain  in  weight  and  water  content  of  dry  corn  and  starch,  when  exposed 
for  different  periods  to  air  saturated  with  moisture.  , 


Days  exposure 

Starch 

Corn 

Per  cent  gain 
in  weight 

Per  cent 
water  content 

Per  cent  gain 
* in  weight 

Per  cent 
water  content 

0 

0 

0 

0 

0 

12.60 

11.19 

8.66 

7.97 

17.30 

14.75 

14.76 

12.86 

20.22 

16.82 

18.58 

15.67 

22.32 

18.24 

21.28 

17.55 

23.86 

19.26 

23.52 

19.04 

25.22 

20.14 

26.00 

20.63 

8 

26.50 

20.95 

27.83 

on  77 

21.69 

9 

28.80 

22.36  1 

31.20 

22.94 

23.78 

30.74 

23.51 

32.41 

24.48 

32.88 

24.74 

34.01 

25.38 

ends  immersed  in  the  water  at  the  bottom.  The  corn  was  weighed 
at  various  intervals  and  the  percentage  water  content  calculated 
from  the  increase  in  weight.  A similar  method  was  used  with 
starch  that  had  been  dried  at  110°  C.,  50  grams  of  the  dry 
starch  being  exposed  in  a wide  crystallizing  dish.  The  room 
temperature  during  the  experiments  was  approximately  20° C. 
The  results  of  the  tests  are  given  in  Table  XIII. 


136  Wisconsin  Research  Bulletin  No.  22 

On  the  fifth  day  and  each  day  afterwards,  when  the  starch 
was  weighed,  it  was  stirred  with  a glass  rod  to  expose  a fresh 
surface.  The  appearance  of  mold  upon  the  starch  brought  the 
experiment  to  a close  on  the  eighteenth  day. 

Results  similar  to  these  were  obtained  by  Rodewald9  by  expo- 
sing between  two  and  three  grams  of  dry  starch  to  a saturated 
air.  The  gain  in  his  experiment  was  somewhat  more  rapid 
than  shown  in  Table  XIII,  because  of  the  relatively  large  surface 
of  starch  exposed  to  the  air.  The  percentage  gain  in  weight, 
and  the  water  content  of  the  starch,  computed  from  his  data, 
are  given  in  Table  XIV. 


TABLE  XIV.  gain  in  weight  of  starch  in  moist  air 

Percentage  gain  in  weight  of  dry  starch,  and  its  water  content  after  exposure  to  air 
saturated  with  water,  for  various  periods. 


Days  of  exposure 

Percent  gain 

Percent  water 

in  weight 

content 

0 

0 

0 

1 

14.55 

12.70 

3 

24.62 

19.75 

28.25  • 

22.03 

11 

32.42 

24.48 

24 

32.54 

24.55 

75 

32.60 

24.59 

The  close  agreement  of  the  results  obtained  in  these  two  in- 
dependent tests,  at  the  eighteen  day  period,  in  spite  of  the  great 
difference  in  the  quantities  of  starch  used,  indicates  that  the 
starch  must  have  beeif  nearly  saturated  with  water  at  this  time. 
This  view  is  confirmed  by  the  fact  that  practically  no  gain  was 
observed  during  the  next  sixty  days  in  Rodewald ’s  experiments. 
At  this  time  the  starch  contained  nearly  25  per  cent  of  water 
which,  calculated  for  the  empirical  formula,  C6H10O5,  of  starch, 
is  equivalent  to  three  molecules  of  water.  This  close  agreement 
to  theory  suggests  that  the  absorbed  water  is  in  molecular  com- 
bination. 

Further  evidence  of  such  a union  is  supplied  by  the  evolution 
of  considerable  heat  when  dry  starch  is  mixed  with  water.  The 
experiments  of  Naegeli10  established  this  fact  and  later  Rode- 

9 Ueber  die  Quellung  der  Staerke.  Landw.  Vers.  Stat.  45,  1895, 

p.  201. 

loTheorie  der  Gaehrung.  1879,  p.  133. 


Role  of  Metabolic  Water  in  Vital  Phenomena  137 

wald11  showed  that  the  heat  evolved  when  dry  starch  is  mixed 
with  water,  at  an  average  temperature  of  about  20° C.  is  equi- 
valent to  23.4  calories  per  gram  of  starch. 

Since  the  water  absorbed  by  starch  is  readily  expelled  at  a 
temperature  of  100 °C.,  the  molecular  combinations  formed  must 
be  feeble;  they  appear  to  be  closely  analogous  to  those  existing 
in  crystals  containing  water  of  crystallization.  The  nature  of 
this  preliminary  combination  differs  widely  from  that  resulting 
when  starch  is  converted  into  dextrose,  in  the  presence  of  dias- 
tase, by  adding  on  to  its  molecule  the  elements  of  water.  In  the 
latter  case,  the  molecule  is  far  more  stable  and  cannot  be  broken 
up  into  starch  and  water  by  the  application  of  heat.  This  dif- 
ference is  also  shown  by  a greater  evolution  of  heat,  when  starch 
is  converted  into  dextrose,  in  spite  of  a much  smaller  quantity 
of  water  being  required  for  the  reaction.  The  theoretical  amount 
of  heat  set  free  in  this  case,  as  well  as  in  other  reactions  occur- 
ring when  one  carbohydrate  is  converted  into  another  in  a plant, 
may  be  calculated  from  the  heats  of  combustion  of  the  substances 
involved. 

The  heats  of  combustion  of  some  of  the  more  important  carbo- 
hydrates found  in,  and  associated  with  the  nutrition  of  plants, 
are  given  herewith.  The  values  selected  are  from  the  tables  of 
Landolt  and  Bornstein  and  are  the  average  of  all  results  obtained 
at  constant  volume.  Complete  oxidation  of  one  gram  of  cellu- 
lose produces  4192.7  gram  calories  (a/  gram,  calorie  being  the 
heat  required  to  raise  the  temperature  of  one  gram  of  water 
from  0°C  to  1°C)  ; of  starch,  4205.2;  of  cane  sugar,  3958.4;  of 
dextrose  and  levulose,  3752.3  gram  calories ; of  water  and  carbon 
dioxide,  none. 

Since  the  thermal  balance  of  all  reactions  that  occur  in  the 
conversion  of  one  substance  into  another  is  always  the  same,  no 
matter  what  course  the  reaction  may  take  or  how  many  inter- 
mediate steps  may  occur,  it  is  possible  to  calculate,  from  the  pre- 
ceding data,  the  heat  equivalent  of  energy  absorbed  or  set  free, 
when  any  one  of  these  carbohydrates  is  changed  into  another. 
Thus  by  the  complete  hydrolysis  of  one  gram  of  starch  there  is 
produced  one  and  one-ninth  grams  of  dextrose.  When  this 
amount  of  dextrose  is  oxidized  to  carbon  dioxide  and  water, 

11  Ueber  die  Quellung  der  Staerke.  Landw.  Vers.  Stat.  45,  1895, 

p.  201. 


138  Wisconsin  Research  Bulletin  No.  22 

there  is  liberated  4169.2  calories;  the  difference  between  this  and 
the  heat  of  combustion  of  the  original  gram  of  starch,  (4205.2), 
is  thirty-six  calories  which  is  the  heat  equivalent  of  the  energy 
set  free  in  the  reaction.  Conversely,  when  one  and  one-ninth 
grams  of  dextrose  are  converted  into  starch,  energy  equivalent 
to  thirty-six  calories  must  be  supplied  from  external  sources, 
if  thermal  equilibrium  is  maintained.  The  energy  balance  be- 
tween any  of  these  carbohydrates  may  be  calculated  in  a similar 
manner. 

The  difference  between  thirty-six  calories,  the  theoretical 
amount  of  heat  evolved  when  one  gram  of  dry  starch  is  convert- 
ed into  dextrose,  and  23.4  calories,  the  heat  evolved,  according 
to  Rodewald,  when  dry  starch  is  mixed  with  cold  water,  amount- 
ing to  12.6  calories  should  represent  the  heat  set  free,  when 
starch  paste  containing  an  equivalent  of  one  gram  of  dry  starch 
is  converted  into  dextrose  by  diastase.  In  order  to  test  this, 
and  thus  independently  to  confirm  the  determination  by  Rode- 
wald. a starch  paste  was  prepared  in  the  usual  manner  from  a 
good  quality  of  corn  starch  by  pouring  boiling  water  over  it 
after  it  had  been  mixed  with  cold  waiter  to  avoid  a lumpy  con- 
dition. 

This  paste  was  strained,  a little  toluol  added  to  prevent  fer- 
mentation, and  placed  in  a constant  temperature  room  for  twen- 
ty-four hours,  when  its  temperature  was  approximately  the 
same  as  the  room.  It  was  thoroughly  mixed  by  pouring  from 
one  vessel  to  another  and  450  c.  c.,  equivalent  to  about  thirty 
grams  of  air-dried  starch,  was  placed  in  each  of  two  silvered 
Dewar  flasks  of  500  c.  c.  capacity.  To  the  contents  of  one  of 
these  flasks,  was  added  50  c.  c.  of  active  malt  extract,  and  to  the 
other  50  c.  c.  of  water,  the  whole  being  carefully  mixed  by  shak- 
ing. A sensitive  thermometer  was  placed  in  each  flask  and  the 
temperature  observed,  at  intervals.  For  forty-eight  hours  the 
temperature  in  both  flasks  remained  between  21.6°  C.  and  21.7° 
C.,  the  temperature  of  the  room  in  the  meantime,  ranging  from 
21.6°  C.  to  21.9°  C.  During  this  period  all  of  the  starch  to 
which  malt  extract  was  added  had  disappeared,  being  convert- 
ed into  dextrose.  The  experiment  was  repeated  a second  time 
with  similar  results,  no  appreciable  change  of  temperature  being 
observed  in  either  test  that  could  be  attributed  to  the  hydrolysis 
of  starch. 


Role  of  Metabolic  Water  in  Vital  Phenomena  139 


Because  of  these  negative  results,  it  seemed  advisable  to  repeat 
the  tests  of  Rodewald,  with  starch  from  the  same  lot  used  in  the 
preceding  experiments,  and  under  similar  conditions.  The 
starch  used  for  this  purpose  lost  9.83  per  cent  in  weight,  when 
dried  at  97°C.  and  10.38  per  cent  at  110°C.  This  coincides 
approximately  with  the  formula  C6H10O5+H2O,  which  is  equiv- 
alent to  a water  content  of  10  per  cent.  An  estimation  of 
starch  in  the  substance  dried  at  110°C.  gave  a purity  of  93.7 
per  cent. 

In  the  first  test  fifty  grams  of  the  air-dried  starch  was  placed 
in  a silvered  Dewar  beaker  and  100  c.  c.  of  water  having  the 
same  temperature  as  the  starch,  added  to  it.  The  starch  and 
water  were  mixed  to  a smooth  paste,  by  stirring  with  a sensi- 
tive thermometer,  and  the  temperature  observed.  The  tempera- 
ture rose  quickly  and  within  five  minutes  reached  a maximum  2° 
C.  higher  than  the  initial  temperature.  This  was  repeated  a 
number  of  times,  with  practically  the  same  results.  Assuming 
the  specific  heat  of  starch  to  be  0.3,  the  heat  liberated  is  equiv- 
alent to  230  calories ; to  this  should  be  added  the  heat  absorbed 
by  the  apparatus,  which,  under  similar  conditions,  was  found 
to  be  approximately  fifteen  calories  for  each  degree  of  change  in 
temperature;  a total,  in  this  case,  of  thirty  calories.  This  makes 
a total  of  260  calories  set  free  when  fifty  grams  of  air  dried 
starch  containing  10  % of  water  was  mixed  with  water,  at  a 
temperature  of  approximately  20°  C.  This  is  equivalent  to  5.2 
calories  per  gram. 

In  a similar  trial  with  starch  that  had  been  exposed  to  air 
saturated  with  moisture,  and  which  contained  25  per  cent  of 
water,  heat  equivalent  to  0.93  calories  per  gram  was  liberated, 
indicating  that  the  starch  used  was  not  entirely  saturated  with 
water. 

The  average  amount  of  heat  liberated,  in  three  trials,  in  which 
fifty  grams  of  starch  dried  at  110°  C.  were  mixed  with  100  c. 
c.  of  water,  was  equivalent  to  21.8  calories  per  gram;  calculated 
for  pure  dry  starch  it  amounts  to  23.3  calories  per  gram,  a 
value  agreeing  closely  with  the  results  obtained  by  Rodewald. 

It  has  been  suggested  that  the  rise  in  temperature,  in  these 
cases)  is  due  to  simple  absorption  and  not  to  a molecular  union 
of  the  starch  and  water ; this  is  unlikely  since  an  equal  amount 
of  dry  filter  paper,  immersed  in  water,  under  the  same  condi- 


140 


Wisconsin  Research  Bulletin  No.  22 


tions,  gave  no  appreciable  change  in  temperature,  although  the 
paper  absorbed  water  more  rapidly  than  did  starch. 

The  foregoing  results  point  directly  to  a feeble  molecular  com- 


bination of  starch  with  water,  analogous  to  that  existing  in 
crystals  containing  water  of  crystallization.  This  is  well  illus- 
trated by  the  behavior  of  calcium  sulphate,  which  crystallizes  as 
gypsum  with  two  molecules  of  water.  Most  of  the  water  of 
crystallization  is  easily  driven  off  at  a temperature  not  much 
above  100°  C.  forming  a white  powder  known  as  plaster  of 
Pans,  which  when  again  mixed  with  water  readily  combines 
with  it  to  form  gypsum  again,  considerable  heat  being  set  free 
by  the  reaction.  If  gypsum  be  heated  to  between  300°  and  400° 
( . instead  of  below  200°  C.,  it  loses  all  of  its  combined  water 


and  is  changed  into  anhydrite,  a form  that  no  longer  combines 
directly  with  water  when  mixed  with  it;  it  still  imbibes  water 
but  no  molecular  combination  occurs  and  no  heat  is  evolved. 
In  this  condition  it  corresponds  to  cellulose  which  is  not  hydro- 
lyzed by  simple  contact  with  water  while  plaster  of  Paris  cor- 
responds to  dry  starch  which  unites  directly  with  water  with  an 
evolution  of  heat,  and  gypsum  itself  corresponds  to  hydrated 
starch  as  it  exists  in  boiled  starch  paste. 


The  heat  liberated,  when  dry  starch  is  mixed  with  wTater,  is 
about  two  thirds  of  the  difference  between  the  heats  of  combus- 
tion of  starch  and  an  equivalent  amount  of  dextrose,  and  it  was 
naturally  expected  that  a further  evolution  of  heat,  sufficient  to 
make  up  this  difference,  would  be  noted  when  starch  paste  was 
converted  into  dextrose  by  diastase.  The  negative  results  ob- 
tained in  tests  with  starch  paste  indicate  either,  that  heat  was 
evolved  in  the  preparation  of  the  starch  paste  and  escaped 
notice  at  the  high  temperature  employed,  or  that  the  hydrolysis 
of  starch  by  diastase  is  associated  with  other  reactions  which 
absorb  sufficient  heat  to  counterbalance  the  heat  evolved  in  hy- 
drolysis. 

Among  changes  of  this  latter  type  is  the  absorption  of  heat 
when  a solid  substance  is  dissolved.  In  this  case,  the  starch  is 
almost  wholly  insoluble  in  water,  while  the  resulting  dextrose 
passes  into  solution  as  rapidly  as  it  is  formed.  The  heat  ab- 
sorbed during  the  solution  of  dextrose  was  determined  by  mak- 
ing the  solution  in  a Dewar  beaker  in  the  same  manner  as  that 
employed  for  obtainihg  the  heat  set  free  when  dry  starch  is 


Role  of  Metabolic  Water  in  Vital  Phenomena  141 

mixed  with  water.  The  preliminary  trials  were  made  with 
commercial  dextrose,  containing  7.83  per  cent  of  water,  which  is 
about  1 per  cent  less  than  the  theoretical  amount  for  one  mole- 
cule of  water  of  crystallization.  Otherwise  the  sample  was  pure. 

In  two  trials,  made  with  this  crystallized  dextrose,  the  heat 
absorbed  during  solution  was  equivalent  to  23.5  and  24.4  calo- 
ries per  gram,  respectively,  an  average  of  23.9  calories  per  gram. 

Two  other  trials  made  with  anhydrous  dextrose  resulted  in 
an  absorption  of  13.25  and  13.27  calories  per  gram  respectively, 
an  average  of  13.26  calories  per  gram.  This  is  at  the  rate  of 
14.7  calories  absorbed  by  tbe  solution  of  the  one  and  one-ninth 
grams  of  anhydrous  dextrose  that  is  derived  from  one  gram  o£ 
pure,  dry  starch. 

These  results  indicate  that  the  solution  of  dextrose  takes  place 
in  two  stages,  the  first  being  a combination  of  the  anhydrous 
substance  with  water  of  crystallization ; this  is  an  exothermic 
reaction  and  heat  is  liberated.  The  second  stage,  in  which 
crystallized  dextrose  is  dissolved,  is  an  endothermic  reaction  the 
heat  absorbed  being  considerably  more  than  the  heat  set  free  in 
the  first  stage.  The  heat  absorbed  when  anhydrous  dextrose 
is  dissolved  in  water  represents  the  difference  between  these 
quantities  whereas  the  total  effect  of  the  endothermic  reaction 
appears  when  crystallized  dextrose  is  dissolved.  The  results  ob- 
tained are  entirely  in  accord  with  those  that  occur  in  the  solu- 
tion of  all  substances  which  unite  with  water  when  they  crystal- 
lize. Thus  anhydrous  calcium  chloride  combines  with  six  mole- 
cules of  water  in  crystallizing  with  an  evolution  of  considerable 
heat;  on  the  other  hand,  the  solution  of  crystallized  calcium 
chloride  absorbs  sufficient  heat  to  act  as  an  efficient  freezing 
mixture.  The  solution  of  substances  like  cane  sugar,  which 
crystallize  without  water,  is  always  accompanied  by  an  absorp- 
tion of  heat. 

The  'thermal  equilibrium  that  prevails  when  starch  paste  is 
hydrolyzed  by  diastase  indicates  that  the  heat  liberated  in  this 
reaction  corresponds  in  quantity  to  the  heat  absorbed  when  the 
resulting  anhydrous  dextrose  is  dissolved  in  water.  If  this  is 
the  case,  the  sum  of  the  calories  set  free,  when  dry  starch  is 
mixed  with  water,  and  the  calories  absorbed  when  an  equivalent 
amount  of  anhydrous  dextrose  is  dissolved  in  water,  should  equal 
the  difference  between  the  heats  of  combustion  of  these  quantities 


142 


Wisconsin  Research  Bulletin  No.  22 


of  dry  starch  and  anhydrous  dextrose.  It  has  been  shown  that 
this  difference,  for  one  gram  of  starch  and  the  dextrose  derived 
from  it  amounts  to  36  gram  calories.  It  was  found  that  the 
heat  liberated  when  dry  starch  is  mixed  with  water,  (23.3  calo- 
ries), added  to  the  heat  absorbed  when  an  equivalent  amount  of 
anhydrous  dextrose  is  dissolved  in  water,  (14.7  calories),  equals 
thirty-eight  calories  per  gram  of  starch,  a difference  of  only  two 
calories  per  gram  between  the  theoretical  value  and  that  found 
by  experiment.  This  value  is  based  upon  the  amount  of  pure 
starch,  found  by  analysis,  in  the  sample  used,  the  effect  of  im- 
purities being;  ignored.  It  is  possible,  however,  that  the  starch 
used  in  these  experiments  may  have  been  as  pure  as  that  used 
m most  calorimetric  tests  upon  which  the  heats  of  combustion 
are  based,  If  this  is  the  case,  the  heat  liberated,  when  water 
is  added,  should  be  calculated  upon  the  total  substance  used  in- 
stead of  upon  the  pure  starch  which  the  sample  contained,  while 
the  heat  absorbed  by  the  solution  of  dextrose  should  be  calculat- 
ed for  an  equivalent  of  the  pure  starch  present.  On  this  basis, 
the  heat  accounted  for,  in  these  experiments  amounts  to  35.5 
calories  per  gram  of  the  starch  used,  which  is  within  half  a 
calorie  of  the  theoretical  amount,  derived  from  the  heats  of 
combustion. 

These  empirical  values  with  starch,  when  considered  in  con- 
nection with  the  heat  evolved  by  air-dry  grain  that  is  immersed 
in  water,  indicate  clearly  the  course  of  the  hydrolysis  of  starch 
in  a germinating  seed  of  corn.  The  first  step  is  the  direct 
union  of  starch  with  water,  the  combination  being  of  the  same 
nature  as  that  in  crystals  containing  water  of  crystallization. 
It  is  this  reaction  which  causes  the  preliminary  rise  in  tempera- 
ture,when  dry  seeds  are  immersed  in  water,  in  the  absence  of 
oxygen.  Then  follows  the  hydrolysis  of  starch  to  dextrose,  by 
diastase,  in  which  the  seed  remains  in  thermal  equilibrium; 
this  change  also  takes  place  in  the  absence  of  oxygen,  and  is 
not  associated  with  the  evolution  of  carbon  dioxide.  The  sub- 
sequent evolution  of  heat,  that  occurs  with  all  varieties  of  seeds, 
when  sprouts  develop,  results  from  the  direct  oxidation  of  dex- 
tiose,  or  of  a similar  carbohydrate,  induced  by  the  respiration  of 
active  cells  situated  in  the  embryo ; it  is  always  associated  with 
an  evolution  of  carbon  dioxide. 

The  direct  union  of  starch  with  water,  in  a feeble  combi- 


Role  of  Metabolic  Water  in  Vital  Phenomena  i43 

nation  analogous  to  water  of  crystallization,  serves  a most  im- 
portant purpose  in  maintaining  a sufficient  amount  of  water  in 
dormant  seeds,  exposed  to  dry  air,  to  insure  the  continued  activ- 
ity of  vital  processes  in  the  few  respiring  cells  of  the  embryo. 
It  is  this  molecular  combination  that  makes  seeds  so  retentive 
of  water,  and  which  enables  germinating  seeds  to  withstand 
drought  without  permanent  injury. 

Reserve  Nutrients  of  Plants 

Unless  each  active  cell  receives  a constant  and  sufficient  sup- 
ply of  available  nutrients  from  external  sources  to  maintain 
respiration  and  other  vital  processes,  the  tissues  of  the  cell  are 
in  part  consumed  and  in  a short  time  the  cell  dies  from  star- 
vation. This  event  is  guarded  against  by  a store  of  reserve 
material  in  each  cell,  which  is  not  used  so  long  asi  available 
nutrients  from  an  external  source  are  supplied,  but  which  may 
be  utilized  to  support  respiration  for  a limited  time,  when  for 
any  reason  the  usual  supply  fails. 

The  primary  source  of  all  organic  nutrients  is  photosynthesis 
which  occurs,  for  the  most  part,  in  the  chlorophyl  bearing  cells 
of  the  leaves,  from  which  the  nutrients  in  a high  state  of  hydra- 
tion are  distributed  by  osmosis  to  all  of  the  active  cells  of  a 
plant.  In  these  latter  cells  the  nutrients  are  in  part  oxidized, 
producing  carbon  dioxide  and  water,  and  in  part  dehydrated. 
Part  of  these  metabolic  products,  as  cellulose,  starch,  fats  etc., 
are  insoluble  and  remain  in  the  cell  either  as  permanent  tissues, 
(cellulose),  or  as  reserve  nutrients,  (starch  and  fats),  to  be 
drawn  upon  whenever  the  supply  from  the  leaves  fails;  other 
paits  are  converted  into  soluble  products,  (cane  sugar,  organic 
acids  etc.,  containing  less  of  the  elements  of  water  than  the 
original  nutrients),  which  are  not  directly  available  as  nutrients 
and  are  excreted  from  the  active  cells  into  the  tracheids  and 
other  vessels  that  transfer  water  from  the  roots,  and  are  carried 
bj  this  current  of  water  to  the  leaves,  where  they  are  again  hy- 
drolized  and  become  available  as  nutrients. 

These  products,  including  even  that  part  of  the  carbon 
dioxide  which  is  dissolved  in  the  sap,  may,  therefore,  be  con- 
sidered to  be  reserve  nutrient  materials  also,  since  the  energy 
required  for  restoring  them  to  an  available  form  is  supplied  by 
photosynthesis,  from  an  external  source.  In  some  plants,  these 


144 


Wisconsin  Research  Bulletin  No.  22 


soluble  products  of  respiration  that  have  been  excreted  from  the 
active  cells  are  the  most  important  of  the  reserve  carbohydrate 
nutrients.  This  is  the  case  with  cane  sugar  in  sugar  cane,  In- 
dian corn,  some  fruits,  and  many  roots  and  tubers  at  the  end 
of  the  first  year’s  growth. 

Some  of  these  reserve  nutrients,  especially  the  insoluble  forms 
such  as  starch,  the  fats  etc.  may  be  hydrolized  by  specific  fer- 
ments in  all  tissues  of  a plant  where  a sufficient  supply  of  water 
is  available.  This  may  also  occur  with  cane  sugar  and  perhaps 
with  other  soluble  carbohydrates,  although  the  change  in  these 
cases  is  more  readily  effected  in  the  leaves,  as  is  shown  by  a 
lower  content  of  sugar  in  the  sap  of  leaves  than  in  that  of  other 
tissues  as  well  as  by  the  rapid  disappearance  of  cane  sugar  from 
beets,  from  bulbs,  and  from  the  sap  of  deciduous  trees,  when 
leaves  appear  in  spring.  The  increase  of  cane  sugar  in  all 
sugar  producing  plants  as  maturity  approaches  and  leaves 
become  less  active,  is  further  evidence  of  this  trend. 

A most  remarkable  thing  about  plant  metabolism  is  that  none 
of  the  organic  excreted  products  of  the  cells,  except  a small  part 
of  the  carbon  dioxide  and  a few  volatile  oils  and  ethers  mani- 
fested by  a characteristic  odor,  are  lost  to  a plant,  after  photo- 
synthesis is  established. 

Water  Produced  During  The  Ripening  of  Fruit 

Some  of  the  best  illustrations  of  the  changes  induced  by  res- 
piration, in  the  character  of  vegetable  tissues,  are  found  in  the 
ripening  of  fruits,  especially  such  as  apples,  pears,  plums,  and 
other  varieties  in  which  the  final  ripening  changes  may  be  nor- 
mally completed  after  the  fruit  is  removed  from  the  tree.  Dur- 
ing the  period  of  growth,  the  stems  of  such  fruits  are  firmly  at- 
tached to  the  parent  branch,  but  as  maturity  approaches,  the 
cellular  structure  in  a portion  of  the  stem  changes,  some  of  the 
cells  being  absorbed  so  that  a slight  strain  may  separate  the 
fruit  from  the  tree.  This  stage  is  usually  reached  while  the 
fruit  is  still  hard  and  green,  and  before  it  has  reached  an  edible 
condition.  It  is  doubtful  if  the  fruit  receives  much  water  or 
other  nutrients  from  the  tree,  after  this  time,  although  its  cells 
are  still  active  and  respiration  continues  at  a rapid,  rate.  Picking 
the  fruit  does  not  stop  the  normal  ripening  changes,  which  pro- 
ceed fully  as  rapidly  after  removal  from  the  tree  as  before. 


Role  of  Metabolic  Water  in  Vital  Phenomena  145 

These  changes  are  manifested  by  an  absorption  of  oxygen,  a 
corresponding  evolution  of  carbon  dioxide,  and  a marked  in- 
crease in  the  mellowness  and  succulence  of  the  fruit. 

During  ripening,  there  is  a constant  loss  of  organic  matter 
through  oxidation,  which  naturally  results  in  the  production  of 
considerable  metabolic  water,  but  the  effect  of  this  upon  the 
succulence  of  the  ripe  fruit  is  to  a considerable  extent  offset  by 
hydrolytic  reactions  in  which  water  is  fixed  in  organic  combi- 
nation. The  tissues  of  green  fruits  nearly  always  contain  starch, 
pectous  substances  and  various  organic  acids  which  mostly  dis- 
appear during  ripening,  being  converted  into  soluble  carbohy- 
drates and  other  compounds  in  which  the  elements  of  water  are 
higher  than  in  the  original  form.  Nearly  all  of  the  changes  oc- 
curring in  the  ripening  of  fruit,  except  those  resulting  from  di- 
rect oxidation,  are  hydrolytic  in  character  and  cause  liquid  water 
to  disappear.  The  water  thus  fixed  in  organic  combination  may 
in  some  cases  exceed  the  metabolic  water  resulting  from  oxida- 
tion and  still  leave  the  ripe  fruit  far  more  succulent  than  the 
green  fruit,  because  more  of  the  organic  matter  of  the  ripe  fruit 
is  dissolved  in  the  fruit  juices. 

It  is  probable,  also,  that  some  water  is  fixed  b}^  photosynthesis, 
when  green  fruits  are  exposed  to  light,  even  after  the  fruits  are 
removed  from  the  tree.  In  addition  to  the  withdrawal  of  water 
for  these  purposes,  there  is  a continual  loss  of  water  into  the  sur- 
rounding air,  even  when  this  is  in  a saturated  condition,  since 
the  carbon  dioxide  evolved  from  the  wet  tissues  must  be  saturat- 
ed with  water. 

In  spite  of  the  water  withdrawn  from  a fruit  in  these  ways, 
during  the  ripening,  there  is,  under  ordinary  conditions,  a con- 
tinual increase  in  the  percentage  of  water  found.  This  increase 
is  partly  due  to  loss  of  organic  matter,  through  oxidation,  and 
partly  to  the  substitution  of  water  for  a portion  of  the  organic 
matter  that  has  disappeared.  It  often  happens,  when  fruit  is 
stored  under  conditions  which  retard  evaporation,  that  the  meta- 
bolic water  produced  by  oxidation,  is  miore  than  sufficient  to  re- 
place that  lost  by  evaporation  and  that  fixed  in  organic  combi- 
nation, in  which  case,  the  absolute  amount,  as  well  as  the  per- 
centage of  water  in  the  ripened  fruit,  exceeds  that  present  in 
the  green  fruit  when  it  was  picked. 


146 


Wisconsin  Research  Bulletin  No.  22 


Relative  Water  Content  of  Green  and  Ripe  Fruit 

During  the  past  four  or  five  years,  as  opportunity  offered,  de- 
terminations of  water  have  been  made  in  various  kinds  of  fruit,' 
at  different  stages  of  maturity,  for  the  purpose  of  ascertaining 
the  amount  and  direction  of  these  changes.  All  of  the  fruits,  ex- 
cept the  apples  and  plums,  were  purchased  in  the  markets  and 
had  been  picked  some  time  when  observations  commenced.  The 
apples  and  plums  were  seedling  varieties  grown  on  the  station 
grounds.  The  fruits  selected  for  each  experiment  were  appar- 
ently in  the  same  stage  of  maturity,  but  with  the  exception  of 
apples  and  plums  may  have  been  grown  on  different  trees,  and 
possibly  in  different  orchards,  under  conditions  which  materially 
affected  the  initial  water  content. 

It  has  been  assumed  in  each  case  that  the  water  content  of 
the  fruit  set  aside  to  ripen  was  the  same  as  that  found  in  the 
green  fruit,  at  the  date  when  this  was  examined,  and  that  the 
difference  in  the  water  content  of  the  green  and  ripe  fruit  is 
the  result  of  ripening  changes.  There  is  some  error  by  this 
assumption,  but  since  in  nearly  every  case  the  results  are  either 
averages  of  several  determinations,  or  are  derived  from  com- 
posite samples  taken  from:  different  specimens,  it  is  believed 
that  they  are  typical  of  the  changes  that  occur  in  the  normal 
ripening  of  fruit. 

The  water  content  was  determined,  in  every  case  by  drying 
to  constant  weight  in  a steam  oven  at  approximately  97° C.  The 
loss  in  weight  was  calculated  on  the  original  weight  of  the  green 
fruit.  The  results  are  given  in  Table  XV. 

The  most  obvious  change,  during  the  ripening  of  fruit,  shown 
m Table  XV,  is  the  increase  in  the  percentage  of  water;  a 
change  that  always  occurs  when  excessive  evaporation  of  water 
is  prevented,  whether  fruit  is  left  hanging  upon  the  tree,  or  is 
picked. 

There  is  a general  impression,  among  fruit  growers,  that 
pears  picked  as  soon  as  the  fruit  will  separate  from  the  branch 
without  breaking  the  stem,  and  stored  in  a dark  place  to  ripen 
are  sweeter  and  more  succulent  than  fruit  left  to  ripen  upon  the 
tree.  This  is  easy  to  understand,  since  the  fruit  receives  little 
water  from  the  tree  after  it  is  mature  and  the  connecting  tissues 
of  the  stem  are  being  absorbed.  It  is  even  doubtful  if  sufficient 


Role  of  Metabolic  Water  in  Vital  Phenomena  147 


TABLE  XV.  WATER  CONTENT  OF  GREEN  AND  RIPE  FRUIT 


Sample  No. 

V ariety 

Green  Fruit 

Ripe  Fruit 

Per  cent 
loss  in 
weight 

Date 

Per  cent 
water 

Date 

Per  cent 
water 

1 

Bartlett  pear 

Aug.  19 

81.64 

Aug.  26 

82.57 

6.08  ' 

9 

Sept.  25 

85.73 

Sept.  30 

86.23 

2 

Bartlett  pear 

Sept.  25 

85.73 

Oct.  4 

87.62 

2.06 

3 

Aug1.  31 

79.76 

Sept.  18 

80.36 

4 

Sept.  9 

77.01 

Sept.  9 

78.02 

5 

Seckel  pear 

Sept.  22  ! 

76.96 

Oct.  1 

78.63 

0.54 

6 

Seckel  pear 

Oct.  2 1 

80.71 

Oct.  15 

81.47 

0.38 

6 

Seckel  pear 

Oct.  2 | 

80.71 

Oct.  20 

81.03 

1.33 

6 

Seckel  pear 

Oct.  2 | 

80.71 

Oct.  22 

81.88 

2.98 

7 

Winter  Nellis  pear 

Nov.  23 

76.52 

Dec  16 

77.95 

0.90 

8 

Winter  Nellis  pear 

Nov.  25 

75.73 

Dec.  8 

77.57 

4.24 

9 

Apples 

Aug.  18 

81.29 

Aug.  31 

83.88 

5.50 

10 

Crab  apples 

Aug.  18 

82.10 

Aug.  31 

79.86 

16.16 

11 

Plums 

Sept.  26 

81.43 

Oct.  5 

82.12 

8.69 

11 

Plums 

Sept.  26 

81.43 

Oct.  6 

82.32 

5.08 

11 

Plums 

Sept.  26 

81.43 

Oct.  6 

82.85 

1.41 

12 

Plums 

Sept.  26 

83.07 

Oct.  5 

83.21 

8.80 

12 

Plums 

Sept.  26 

83.07 

Oct.  6 

83.65 

5.95 

13 

Plums 

Sept.  26 

79.67 

Oct.  13 

78.69 

11.54 

13 

Plums 

Sept.  26 

79.67 

Oct.  21 

79.76 

.8.75 

14 

Plums 

Sept.  3 

87.00 

Sept.  7 

87.89 

1.40 

14 

Plums 

Sept.  3 

87.00 

Sept.  13 

89.49 

4.36 

15 

Plums 

80.67 

Sept.  13 

82.10 

6.12 

15 

Plums 

80.67 

Sept.  6 

80.80 

15 

Plums 

Sept.  3 

80.67 

Sept.  14 

81.75 

15 

Plums 

Sept.  3 

80.67 

Sept.  18 

81.90 

15 

Plums 

Sept.  3 

80.67 

Sept.  24 

83.84 

16 

Plums 

Sept.  18 

81.90 

Sept.  20 

82.68 

0.96 

16 

Plums 

Sept.  IS 

81.90 

Sept.  22 

83.60 

2.42 

16 

Plums 

Sept.  18 

81.90 

Sept.  24 

83.82 

L82 

ir 

Plums 

Sept.  24 

83.84 

Sept.  29 

84.96 

3.24 

IT 

Plums 

Sept.  24 

83.84 

Oct.  4 

84.84 

9.93 

18 

Plums,  pulp  only,  same  as  No.14 

Sept.  7 

90.49  • 

18 

Plums,  pulp  only,  same  as  No.14 

Sept.  13 

91.55 

19 

Pulp  from  No.  15 

Sept.  6 

86.06 

Sept.  13 

86.35 

19 

Pulp  from  No.  15 

Sept.  6 

86.06 

Sept.  14 

86.75 

19 

Pulp  from  No.  15 

86.06 

Sept.  18 

86.72 

19 

Pulp  from  No. 15 

Sept.  6 

86.06 

Sept.  20 

87.37 

19 

Pulp  from  No.  15 

Sept.  6 

86.06 

Sept.  22 

87.99 

19 

Pulp  from  No.  15 

Sept.  6 

86.06 

Sept.  24 

88.25 

19 

Pulp  from  No.  15 

Sept.  6 

86.06 

Sept.  24 

88.37 

19 

Pulp  from  No.  15 

Sept.  6 

86.06 

Sept.  29 

90.81 

19 

Pulp  from  No.  15' 

Sept.  6 

86.06 

Oct.  4 

88.50 

20 

.1  apanese  persimmon 

Oct.  28 

86.06 

Nov.  6 

75.17 

4.22 

20 

Japanese  persimmon 

1 Oct.  28 

86.06 

Nov,  19 

79.68 

0.81 

21 

Japanese  persimmon  

Oct.  30 

76.75 

Nov.  18 

77.43 

1.11 

21 

Japanese  persimmon 

Oct.  30 

86.06 

Nov.  16 

77.38 

0.79 

22 

Mushroom,  (coprinus) 

Sept.  13 

95.16 

Sept.  14 

95.51 

2.29 

23 

Mushroom,  (coprinus) 

Sept.  21 

93.13 

Sept.  22 

94.42 

3.42 

148 


Wisconsin  Research  Bulletin  No.  22 


water  is  derived  from  the  tree  during  this  period  to  replace 
that  lost  by  evaporation  which,  on  account  of  exposure  to  free 
circulation  of  air,  is  much  greater  than  with  fruit  stored  in 
bulk.  It  is  probable  that  so  long  as  green  fruit  is  exposed  to 
light,  photosynthesis  with  the  production  of  a carbohydrate  from 
carbon  dioxide  absorbed,  and  water  abstracted  from  the  fruit, 
continues  to  occur  and  thus  diminish  the  percentage  of  water 
in  fruit  ripened  upon  the  tree.  It  is  not  unlikely  that  the  mealy, 
granular  condition  often  found  in  such  fruit  is  partly  due  to 
this  cause.  No  doubt  such  changes  also  take  place  after  fruit 
is  gathered,  if  it  is  exposed  freely  to  light,  and  tend  to  make 
the  fruit  less  succulent  than  when  it  is  stored  in  darkness. 

Succulence  of  fruit  depends,  not  only  on  the  ratio  between 
the  solids  and  water,  but  on  the  nature  of  the  solids.  A fruit 
containing  much  of  such  solids  as  sugar  and  organic  acids  that 
are  soluble,  appears  to  be  much  more  succulent  than  a fruit  in 
which  the  organic  matter  is  chiefly  in  the  form  of  cellulose, 
starch,  and  insoluble  pectous  substances,  which  usually  predom- 
inate in  green  fruit.  During  the  ripening  changes  these  in- 
soluble substances  mostly  disappear  being  replaced  by  soluble 
carbohydrates,  a change  which  makes  the  water  content  appear 
to  increase,  even  when  an  actual  decrease  has  occurred. 

The  loss  in  weight,  through  respiration  and  transpiration, 
during  ripening,  is  not  necessarily  associated  with  a shrinkage 
m volume  since  the  products  of  oxidation  of  organic  matter, 
aside  from  carbon  dioxide,  are  usually  of  about  the  same  and 
often  of  greater  volume  than  the  substances  that  have  disappear- 
ed; this  is  especially  true  when  fruits  are  ripened  in  a nearly 
saturated  air,  where  evaporation  is  small.  It  is  chiefly  for  this 
reason  that  gathered  fruits  sweat  when  placed  in  large  piles  or 
m close  receptacles,  the  exudation  being  greatly  increased  by 
internal  pressure  induced  by  an  increase  in  the  volume  of  the 
products  formed. 

With  some  fruits  that  have  a tender  skin,  the  internal  pres- 
sure is  manifested  by  exudations  of  juice  upon  the  surface,  or 
by  cracking  of  the  skin.  It  frequently  happens  during  damp 
weather  that  plums  crack  upon  the  tree.  That  this  is  not 
caused  by  water  derived  from  the  tree  is  evident,  since  sound 
fruit  placed  in  a covered  vessel  in  which  the  air  is  nearly  satur- 
ated with  moisture  cracks  fully  as  much  as  fruit  left  upon 
the  tree. 


Role  of  Metabolic  Water  in  Vital  Phenomena  149 

The  influence  of  free  evaporation  and  possibly  of  photosyn- 
thesis is  clearly  shown,  in  the  case  of  plums,  in  experiment  15 
of  Table  XV  where  the  first  sample,  picked  September  3,  con- 
tained 80.67  per  cent  water.  The  ripe  sample  analyzed  Septem- 
ber 13  was  picked  the  same  day  as  the  first  and  allowed  to  ripen 
in  a covered  vessel  in  the  laboratory.  The  ripe  sample  con- 
tained 82.10  per  cent  water,  an  increase  of  1.43  per  cent  in  ten 
days.  All  of  the  other  samples  were  picked  on  the  date  when 
the  analyses  were  made.  In  every  case  there  was  found  a 
higher  percentage  of  water  than  on  the  first  date  but  with  the 
exception  of  the  last  sample  picked  September  24  when  the 
fruit  was  overripe  and  past  its  best  condition,  none  of  them 
contained  so  high  a percentage  of  water  as  did  the  sample  ripen- 
ed off  the  tree. 

Intramolecular  Respiration  of  Fruits 

The  fleshy  tissues  of  fruit  consist  largely  of  living  cells  which 
continue  to  be  active  for  some  time  after  the  fruit  is  mature, 
even  though  it  may  be  separated  from,  the  parent  plant.  The 
ripening  changes  of  the  fruit,  by  which  its  texture  is  broken 
down,  its  succulence  increased,  and  its  characteristic  flavors 
developed,  are  all  vital  processes  depending  upon  these  cells 
and  anything  that  interferes  with  their  activity  affects  the 
quality  of  the  ripened  fruit.  If,  at  any  stage  of  ripening,  the 
cells  are  all  killed,  the  process  is  suspended,  except  for  slight 
changes  induced  by  specific  enzymes  present  in  the  fruit.  If 
the  cells  are  killed  by  heat,  or  by  poisons  that  suspend  the  act- 
ivity of  enzymes,  the  fruit  remains  indefinitely  in  the  same 
condition.  All  methods  employed  to  preserve  fruit  involve 
this  principle  as  well  as  the  exclusion  or  inhibition  of  the  or- 
ganisms of  decay. 

Energy  required  for  maintaining  the  activity  of  fruit  cells 
arises  from  the  oxidation  of  organic  nutrients  in  the  same  way 
as  in  other  tissues  of  a plant.  Oxygen  required  for  this  may 
be  derived  from  the  air  by  direct  respiration,  as  in  normal 
ripening,  or  energy  may  be  derived  from  organic  nutrients  con- 
taining oxygen,  by  intramolecular  respiration.  The  amount  of 
nutrients  required  in  the  latter  case,  to  supply  the  necessary 
energy,  is  greater  than  when  free  , oxygen  is  available,  since  the 
substances  formed  in  intramolecular  respiration  are  not  com- 


150 


Wisconsin  Research  Bulletin  No.  22 


pletely  oxidized.  Among  these  products  are  various  organic 
acids,  alcohols,  aldehydes,  and  esters,  which  are  more  or  less 
toxic  to  living  cells,  especially  when  abundant.  Moreover  these 
products  accumulate  in  the  fruit  rapidly,  since  no  means  are 
provided  for  their  removal  and  restoration  into  suitable  nu- 
trients, as  is  the  case  in  a growing  plant  through  photosyn- 
thesis. For  these  reasons,  the  life  of  cells  composing  the  tissues 
of  fruit,  from  which  free  oxygen  is  excluded,  is  relatively  short, 
and  the  resulting  texture  and  flavor  of  the  fruit  are  widely  dif- 
ferent from  that  developed  when  the  fruit  is  ripened  in  con- 
tact with  air.  This  has  been  illustrated  in  several  experiments 
with  apples  and  pears,  during  the  past  ten  years.  Both  of  these 
fruits,  if  picked  at  early  maturity  and  placed  in  oxygen-free 
air,  continue  to  evolve  carbon  dioxide  for  three  or  four  weeks, 
during  which  time  the  general  appearance  of  the  fruit  remains 
practically  unchanged.  Its  cells,  however,  die  and  it  acquires 
an  unnatural  odor  and  taste  suggestive  of  corn  silage.  When 
exposed  to  air,  these  dead  fruits  dry  quickly  and  a cut  surface 
remains  white  indefinitely,  in  sharp  contrast  with  the  behavior 
of  fruits  of  the  same  variety  that  have  been  ripened  in  normal 
air.  The  water  content  of  fruit  deprived  of  free  oxygen  in- 
creases considerably  during  the  first  few  weeks  but,  because  of 
its  peculiar  texture,  the  succulence  of  the  fruit  appears  to  be 
no  further  advanced  than  in  the  green  fruit,  at  the  beginning. 

If  oxygen  be  excluded  from  these  fruits  for  a longer  period 
then  three  or  four  weeks,  the  color  of  the  skin  gradually  changes 
to  dull  brown,  the  cut  surface  also  becomes  dark  and  appears 
to  be  water  soaked.  Drops  of  exuded  water  appear  upon  the 
surface,  and  sometimes  water  collects  in  the  bottom  of  the  ves- 
sel. The  acidity  of  the  fruit  increases  and  the  acid  odor  is 
more  marked.  The  texture  remains  unchanged.  After  eight 
or  ten  months,  no  further  change  is  apparent,  so  long  as  free 
oxygen  is  excluded. 

It  seems  probable  that  all  vital  activity  of  the  fruit  cells  ceases 
when  no  more  carbon  dioxide  is  evolved,  since  this  indicates 
the  total  suspension  of  intramolecular  respiration.  In  no  case 
has  this  continued,  with  either  apples  or  pears,  beyond  thirty 
days.  The  changes  that  occur  after  this  time  appear  to  be 
caused  by  enzymes  present  in  the  fruits,  since  the  action  of 
these  is  not  dependent  upon  vital  processes.  In  a few  cases, 


Role  of  Metabolic  Water  in  Vital  Phenomena  151 


anaerobic  organisms  have  attacked  the  fruit  from  which  oxygen 
was  excluded,  causing  it  to  decay  rapidly,  but  changes  of  this 
nature  are  easily  distinguished  from  those  induced  by  active 
cells  of  the  fruit. 

No  difference  has  been  noticed  in  the  final  results  when  oxygen 
has  been  replaced  by  hydrogen,  nitrogen,  carbon  dioxide,  or  by 
th e residual  gases  when  the  oxygen  in  the  containing  vessel  is 
absorbed  by  the  direct  respiration  of  the  fruit  itself.  In  the 
latter  case,  active  intramolecular  respiration  has  been  delayed 
until  the  free  oxygen  was  exhausted.  The  same  results  were 
obtained  when  oxygen  was  excluded  by  immersing  the  fruit  in 
cotton  seed  oil.  This  method  has  some  advantages,  since  it 
prevents  the  passage  of  anaerobic  organisms  from  one  specimen 
to  another. 

Table  XVI  shows  the  loss  in  weight  and  the  water  content  of 
Seckel  pears  that  were  kept  in  carbon  dioxide. 


table  xvi.  water  content  of  pears  in  carbon  dioxide 

Water  content  and  loss  in  weight  of  Seckel  pears  kept  in  carbon  dioxide. 


Months  exposed 

Per  cent,  water  i 
content. 

1 

Per  cent  loss  in  1 
weight. 

Per  cent  of 
dry  matter 
referred  to 
fresh  fruit 

80.71 

85.88 

85.81 

19.29 

14.27 

18.33 



2.87 

5.87 

Changes  in  fruits,  when  deprived  of  free  oxygen  are  analog- 
ous to  those  found  in  similar  experiments  made  in  cooperation 
with  Dr.  Russell,12  with  succulent  plants,  to  determine  the 
causes  operative  in  the  production  of  silage. 

The  succulent  tissues  of  growing  plants  of  all  kinds  behave 
in  an  analogous  manner,  when  deprived  of  free  oxygen,  the  only 
difference  arising  from  the  relative  numbers  of  active  cells  pres- 
ent and  the  amount  of  suitable  nutrients  available  for  the  sup- 
port of  intramolecular  respiration.  In  young  succuiem  tissues, 
in  which  the  cells  are  active  and  the  nutrients  mostly  in  a solu- 
ble form,  that  is  directly  available,  the  production  of  acid  pro- 
ducts is  rapid  and  the  life  of  the  cells  relatively  short.  Plants 
in  this  condition  are  not  suited  for  silage,  since  they  produce  an 

1?  17th  Rpt.  Wis.  Exp.  Sta.,  1900,  p.  123. 

18th  Rpt.  Wis.  Exp.  Sta.,  1901,  p.  177. 


152  Wisconsin  Research  Bulletin  No.  22 

excessively  wet,  acid  product  that  has  little  nutritive  value  On 
the  other  hand,  the  same  variety  of  plant,  in  a more  mature  con- 
dition, with  comparatively  few  active  cells,  and  with  most  of 
ie  nutrients  in  an  insoluble  form,  makes  an  excellent  silage  con- 
taining a minimum  amount  of  water  and  acid,  and  having  a 
ugh  nutritive  value.  The  prevailing  practice,  among  the  bet- 
ter farmers  is  fully  in  accord  with  these  principles. 


Movement  of  Water  in  Plants 
No  question  has  been  raised  by  any  one  regarding  the  prim- 
ary source  of  water  used  by  plants,  but  there  has  been,  and 
S 1 !'S'  great  difference  of  opinion  regarding  the  method  of  its 

distribution  in  the  plant.  A full  account  of  the  experimental 
"oi  earing  upon  the  problem  has  been  given  by  Pfeffer18 
also  by  H.  Marshall  Ward.14  Extended  and  careful  observa- 
tions regarding  phenomena  associated  with  the  flow  and  pressure 
of  sap  in  the  maple  were  made  by  Jones,  Edson  and  Morse.13  A 
more  recent  resume  of  . the  subject,  especially  in  relation  to  the 
action  of  living  cells  upon  transpiration  and  sap  flow  has  been 
made  by  Overton.16 

It  is  conceded  by  all  that  water,  absorbed  by  the  root  hairs 
passes  up  through  the  sap  wood,  which  abounds  in  active  cells,’ 
to  the  leaves,  where  it  is  either  used  in  photosynthesis  for  the 
production  of  organic  matter,  or  passes  into  the  air  by  transpi- 
ration. Little  if  any  water  is  transmitted  through  the  heart 
wood,  which  contains  no  living  protoplasm.  A tree  may  con- 
tinue to  grow,  and  maintain  all  of  its  normal  functions,  for  an 
indefinite,  period,  after  the  heart  wood  is  wholly  destroyed  by 
fungus  disease.  This  suggests  a close  causal  relation  between 
the  vital  processes  occurring  in  the  sap  wood  and  the  transmis- 
sion  of  water  to  the  leaves. 

One  of  the  most  characteristic  functions  of  living  protoplasm 
is  respiration  which  is  always  manifested  by  an  evolution  of 
carbon  dioxide.  In  most  cases  it  is  also  associated  with  a direct 
absorption  of  free  oxygen  although,  if  suitable  nutrients  are 
continually  supplied  and  the  waste  products  removed  as  fast 


13  Physiology  of  Plants. 

14  Timber  and  Some  of  Its  Diseases 
45  Bulletin  103,  Vt.  Exp.  Sta. 

Studies  on  the  Relation  of  the  Living  Cell  to  the 
gap  Flow  ip  Pyperps,  Bot.  Qf«.  ,Tan.  and  Feb. 


Transpira,tiop  anfl 


Role  of  Metabolic  Water  in  Vital  Phenomena  153 

as  they  are  formed,  respiration  may  for  some  time  be  wholly 
intramolecular,  no  free  oxygen  being  removed.  The  cells  of 
living  tissue  are  continually  respiring  by  one  or  both  of  these 
methods  and  in  consequence  there  is  a constant  production  of 
carbon  dioxide,  water,  and  certain  soluble  organic  compounds 
in  these  tissues.  The  corky  tissues  of  the  bark  interpose  an  al- 
most impervious  barrier  to  the  direct  escape  of  the  carbon  di- 
oxide formed.  On  the  other  hand,  the  sap  wood  with  its  porous 
structure,  through  which  a current  of  water  is  continually  pass- 
ing, provides  an  efficient  means  for  its  removal,  since  carbon 
dioxide,  especially  when  subjected  to  pressure,  is  readily  solu- 
ble in  water  and  is  either  carried  by  it  in  solution,  or  passes 
by  osmosis  from  one  vessel  to  another  to  the  leaves  where  it  is 
restored  by  photosynthesis  into  nutrient  materials  available  for 
the  support  of  respiration.  The  amount  of  carbon  dioxide  in 
the  gases  dissolved  in  the  sap  of  growing  plants,  which  amounts 
to  100  to  500  times  that  in  normal  air,  and  from  ten  to  fifty 
times  that  in  soil  air,  confirms  the  view  that  this  is  the  usual 
path  by  which  the  gaseous  products  of  respiration  are  removed. 

The  presence  of  organic  acids,  sugars  of  various  kinds,  and 
other  products  formed  by  protoplasmic  activity,  in  the  sap  is 
further  evidence  of  a direct  transfer  of  soluble  matter  from  the 
active  cells  to  the  vessels  in  which  water  is  carried  to  the  leaves, 
since  the  water  absorbed  by  the  roots  contains  none  of  these 
substances. 

For  the  most  part,  organic  substances  found  in  circulating 
sap  are  products  of  intramolecular  respiration  and  do  not  serve 
as  direct  nutrients  for  growing  cells.  Some  of  these  products 
may  be  converted  into  suitable  nutrients  by  specific  enzymes  in 
any  part  of  a plant  where  direct  respiration  occurs,  others  are 
made  available  as  nutrients,  only  by  photosynthesis,  chiefly  in 
the  chlorophyl  bearing  cells  of  the  leaves. 

It  is  highly  probable  that  the  evolution  of  carbon  dioxide 
during  respiration,  is  one  of  the  chief  factors  affecting  sap  flow 
and  pressure  in  growing  plants.  Sachs  advanced  the  idea  that 
the  change  in  volume  of  gases  in  plant  tissues,  caused  by  varia- 
tion of  temperature  is  the  cause  of  changes  in  sap  pressure,  and 
through  its  intermittent  action,  of  sap  movement.  This  view 
is  discussed  by  Jones,  Edson  and  Morse 17  In  its  relation  to 


it  Loc.  cit. 


Wisconsin  Research  Bulletin  No.  22 


.154 


phenomena  observed  in  the  maple  and  other  trees,  in  which 
pressures  occur  many  times  greater  than  can  he  accounted  for 
by  simple  expansion  at  ohserved  temperatures,  and  while  admit- 
ting that  this  factor  has  some  influence,  they  consider  it  to  be 
of  minor  importance. 

There  is,  however,  one  phase  of  this  question  that  has  ap- 
parently been  overlooked  and  that  is  the  influence  which  tem- 
perature and  pressure  have  upon  the  solubility  of  carbon  dioxide 
m water.  The  behavior  of  carbon  dioxide,  in  this  respect,  differs 
greatly  from  that  of  either  oxygen  or  nitrogen,  the  gases  with 
which  it  is  associated  in  the  air.  For  comparison,  the  solubili- 
ties of  nitrogen,  oxygen  and  carbon  dioxide,  in  water,  are  given 
in  Table  XVII.  The  values  for  each  gas  are  taken  from  A. 
Seidell ’s  tables  of  solubilities. 


TABLE  XVII.  SOLUBILITY  OF  NITROGEN,  OXYGEN,  CARBON  DIOXIDE 

to  oSne  cSOlcVeof^ at  <Merent  ,e™tTirtures.  The  wei(rhts  and  TOlumes  are 


Temperature 


Nitrogen 

Oxygen 

Carbon 

Dioxide 

1 Wt.  g-ms. 

Vol.  c.  c. 

Wt.  gins. 

Vol.  c.  c. 

1 Wt.  g-ms. 

| Vol.  c.  c. 

.00239 

.0235 

.00695 

.0489  1 

.335 

1.713 

, 00259 

.0208 

.00607 

.0429 

.277 

1.424 

.00230 

.0186 

.00537 

.0380  | 

.231 

1 .194 

.00208 

.0179 

.00480 

.0342 

.197 

1 .019 

.00189 

.0164 

.00434 

.0310  ; 

.169 

0.878 

.00174 

.0150 

.00393 

.0283 

.145 

.759 

.00161 

.0138 

.00359 

.0261 

.126 

.665 

It  appears  from  Table  XVII  that,  at  0°C.,  water  dissolves  1.7 
times  its  volume  of  carbon  dioxide  which  is  thirty-five  times 
greater  than  the  volume  of  oxygen  and  more  than  seventy 
times  greater  than  the  volume  of  nitrogen  dissolved  at  the  same 
temperature.  It  is  also  evident  that  the  solubility  of  carbon 
dioxide  increases  more  rapidly  as  temperature  falls  than  either 
Df  the  other  gases,  the  increase  in  solubility  for  one  degree 
change  in  temperature  between  0°  and  5°C.  being  more  than  the 
total  solubility  of  either  nitrogen  or  oxygen  at  0°C.  It  is  ob- 
vious that  while  it  may  be  permissible  to  ignore  the  influence  of 
solubility  upon  the  pressures  of  both  oxygen  and  nitrogen,  in 
the  presence, of  water,  this  cannot  be  done  without  serious  error 
if  a large  proportion  of  carbon  dioxide  is  present,  as  is  always 
the  case  in  the  gas  in  plant  tissues,  especially  when  photosvnthe- 


Role  of  Metabolic  Water  in  Vital  Phenomena 


155 


sis  is  suspended,  at  which  times,  maximum  pressures  usually 
occur. 

No  data  are  available  regarding  the  solubility  of  carbon  di- 
oxide at  temperatures  below  0°C.,  but  since  its  solubility  in- 
creases as  this  point  is  approached,  it  is  probable  that  the  sol- 
ubility increases  still  more  rapidly  as  the  temperature  falls  be- 
low freezing.  This  has  a direct  bearing  upon  the  problem  since 
the  sap  of  plants  always  contains  substances  (chiefly  sugars  and 
organic  acids)  in  solution  which  naturally  lower  its  freezing 
point.  Moreover,  as  freezing  proceeds  the  concentration  of  the 
sap  increases  causing  the  freezing  point  i;->  be  continually  lower- 
ed. It  seems  probable  therefore,  that  some  concentrated  sap 
always  remains  liquid  at  all  temperatures  which  prevail  during 
the  season  when  sap  flows  or  when  sap  pressure  is  observed. 
The  negative  pressure  observed  at  low  temperatures,  as  well  as 
the  positive  pressure  that  follows  when  the  temperature  rises 
above  freezing,  are  in  strict  harmony  with  the  view  that  carbon 
dioxide  is  dissolved  at  the  lower  temperature  and  liberated  from 
solution  when  the  temperature  is  raised. 

Sap  pressure  in  maple  and  certain  other  trees  is  most  pro- 
nounced in  early  spring,  before  the  appearance  of  leaves,  when 
transpiration  is  slow  and  when  the  diurnal  range  in  temperature 
is  wide  and  when  respiration  is  stimulated  by  an  abundance  of 
suitable  nutrients,  in  soluble  form,  that  have  accumulated  dur: 
ing  a prolonged  dormant  period.  The  conditions  are  therefore 
most  favorable  for  the  production  of  a maximum  pressure  during 
the  day  and  of  a minimum,  perhaps  a negative  pressure  during 
the  colder  nights.  Among  the  most  important  factors  that  con- 
tribute to  these  results  is  the  influence  of  temperature  upon  the 
evolution  and  solubility  of  carbon  dioxide  and  this  alone  is  be- 
lieved to  be  sufficient  to  explain  sap  pressure  and  the  bleeding  of 
the  maple  and  other  plants  which  exhibit  these  phenomena  in 
early  spring,  before  photosynthesis  is  established.  It  certainly  is 
not  dependent  on  water  derived  directly  from  roots,  for  trees  cut 
in  early  winter  before  any  sap  pressure  has  developed  bleed  copi- 
ously in  the  warm  days  of  spring  and  under  favorable  conditions 
the  buds  swell  and  may  open.  Nursery  stock  kept  in  storage  dur- 
ing the  winter  under  conditions  that  allow  no  absorption  of  water 
through  the  roots,  put  forth  leaves  and  blossoms  in  the  spring. 
The  same  is  true  of  most  bulbs  and  tubers  which  send  forth 
long  sprouts  with  no  external  water  supply. 


156 


Wisconsin  Research  Bulletin  No.  22 


As  the  season  advances,  the  temperature  rises  and  becomes 
more  uniform.  These  conditions  favor  a more  rapid  respiration 
with  a greater  evolution  of  carbon  dioxide  and  if  other  condi- 
tions remained  the  same,  an  enormous  pressure  would  necessarily 
result  from  the  greatly  increased  vital  activity.  Coincident 
with  these  changes  leaves  develop,  photosynthesis  is  established, 
transpiration  increases,  and  the  pressure  is  relieved.  A large 
part  of  the  carbon  dioxide  resulting  from  respiration,  and  much 
water,  are  utilized  during  photosynthesis,  in  the  production  of 
organic  nutrients  and  a large  amount  of  water  escapes  through 
the  leaves  in  transpiration.  These  functions  not  only  prevent 
excessive  pressure  but  usually  induce  a negative  pressure  dur- 
ing the  growing  season. 

During  the  growth  of  an  annual  plant  200  to  400  pounds 
water  are  absorbed  by  roots  and  transpired,  for  each  pound  of 
dry  matter  contained  in  the  mature  plant.  Since  the  elements 
of  water  comprise  but  little  more  than  half  the  weight  of  the 
dry  matter  of  plants,  this  amount  is  greatly  in  excess  of  the  ac- 
tual needs  for  growth,  but  in  addition  to  supplying  material  for 
building  tissue,  water  is  the  medium  by  which  the  waste  pro- 
ducts of  respiration  are  removed  from  the  cells  and  carried  to 
the  leaves  for  restoration  into  nutrients  that  may  be  used 
again.  A large  amount  of  water  is  required  for  this  function 
because  the  concentration  of  the  excreted  products  must  be 
kept  low  to  avoid  injury,  and  in  the  case  of  carbon  dioxide, 
which  is  but  slightly  soluble,  to  insure  its  solution  and  prevent 
its  waste. 

During  the  day,  when  photosynthesis  is  active,  the  proportion 
of  carbon  dioxide  in  the  gas  of  plants  is  lower  than  in  early 
morning,  after  the  plant  has  been  in  darkness  for  several  hours. 
This  indicates  that  the  carbon  dioxide  liberated  b}^  respiration 
during  the  shaded  period  has  not  escaped  through  the  leaves 
and  bark  as  rapidly  as  it  has  been  formed,  but  has  been  dissolved 
For  the  most  part,  in  the  plant  sap,  and  held  in  reserve  for  resyn- 
thesis into  available  nutrients  when  day-light  returns.  This 
principle  applies  also  to  other  soluble  products  of  respiration 
found  in  the  circulating  sap  of  plants,  most  of  which  are  return- 
ed to  the  leaves  and  converted  into  suitable  nutrients  to  be  used 
again.  The  carbon  dioxide  evolved  by  a plant  in  darkness  is  not 
therefore  an  accurate  measure  of  its  vital  activity,  unless  the 


Role  of  Metabolic  Water  in  Vital  Phenomena  157 


shaded  period  is  quite  prolonged,  since  a large  part  of  the  carbon 
dioxide  formed  under  these  conditions  is  held  in  solution  in  the 
sap,  and  finally  converted  into  suitable  nutrients  for  the  plant. 

This  retention  of  carbon  dioxide  and  other  products  of  res- 
piration, when  a plant  is  shaded,  serves  a most  important  pur- 
pose in  the  conservation  of  carbohydrate  nutrients,  which  must 
otherwise  be  lost,  since  during  photosynthesis  these  excreted 
products  are  restored  to  their  original  form  and  once  more  made 
available  for  the  building  of  tissue  and  fo,r  maintaining  the  vital 
forces.  By  this  means  the  waste  of  nutrients  through  respira- 
tion is  greatly  reduced  and  the  rate  of  growth  correspondingly 
increased.  In  this  respect  chlorophyl  producing  plants  are  far 
more  economical  in  the  utilization  of  nutrients,  than  are  sapro- 
phytic plants  or  animals,  which  have  no  provision  for  restoring 
products  of  respiration  into  nutrients  that  may  be  used  again 
for  maintenance  or  growth,  and  which  must  expend  considerable 
energy  in  the  elimination  of  these  useless  and  often  poisonous 
excretions. 

The  behavior  of  girdled  trees  and  plants  shows  that  water 
absorbed  by  roots  is  transferred  in  some  way,  through  the  woody 
tissue  to  the  leaves  and  that  very  little  movement  of  water,  in 
this  direction,  occurs  through  the  more  active  cells  of  the  cam- 
bium and  cortex.  No  doubt  these  derive  some  water,  by  osmosis, 
directly  from  the  channels  through  which  the  upward  current 
of  water  is  carried,  but  most  of  the  water  in  these  tissues  appears 
to  pass  first  to  the  leaves  and  from  there  in  organic  combina- 
tion, to  the  growing  cells,  in  all  parts  of  the  plant. 

It  is  well  established  that  organic  nutrients  are  synthesized 
in  the  leaves  and  then  distributed.  The  course  taken  by  these 
nutrients  cannot  be  through  the  same  channels  by  which  water 
is  brought  from  the  roots  to  the  leaves,  for  the  current  in  these 
vessels  is  always  in  the  opposite  direction,  and  during  active 
transpiration,  is  far  too  rapid  for  osmosis  to  overcome.  Since 
there  is  no  free  current  of  water  through  the  growing  cells  these 
nutrients  must  pass  from  one  active  cell  to  another,  by 
osmosis,  from  the  leaves  to  the  remotest  part  of  a plant.  Every 
condition,  in  a growing  plant,  is  favorable  for  such  a movement , 
the  concentration  of  nutrients  being  naturally  higher  in  the 
leaves,  where  nutrients  are  elaborated,  and  where  evaporation 
is  most  rapid,  than  in  neighboring  cells  where  no  synthesis  oc- 


Wisconsin  Research  Bulletin  No.  22 


1 58 

curs  and  from  which  respiration  is  constantly  removing  a por- 
tion of  the  dissolved  substance  and  replacing  it  in  part,  with 
metabolic*  water.  Moreover  this  condition  holds  for  each  suc- 
ceeding cell  wherever  active  protoplasm  exists.  There  is  of  : 
course  a transfer  of  water,  by  osmosis,  in  the  opposite  direction, 
from  the  growing  cells  towards  the  leaves  but  this  is  more  than 
compensated  by  the  production  of  metabolic  water  through  the 
oxidation  and  dehydration  of  organic  nutrients.  When  they 
start  from  the  leaf,  carbohydrate  nutrients  are  in  the  form  of 
dextrose  or  similar  substance  containing  a large  proportion  of  the 
elements  of  water,  but,  as  they  proceed,  a portion  is  completely 
oxidized  by  the  respiration  of  each  cell  and  all  of  its  potential 
water  set  free ; a portion  is  deposited  as  cellulose  or  starch  con- 
taining less  of  the  elements  of  water  than  the  original  nutrient ; 
and  still  other  portions  are  transferred,  together  with  various 
products  of  respiration,  into  the  current  of  water  that  flows 
constantly  through  the  woody  tissues  from  the  roots,  and  N 
are  carried  back  to  the  leaves  from  which  point  the  cycle  is 
repeated,  so  long  as  water  is  supplied  to  the  roots  and  conditions 
are  favorable  for  the  synthesis  of  organic  nutrients  in  the  leaves,  i 

One  of  the  most  important  results  of  this  cycle  is  the  libera- 
tion of  metabolic  water  in  all  growing  cells.  It  is  to  this  that 
the  high  water  content  of  such  tissue  is  chiefly  due  and  it  is  this 
more  than  any  other  factor,  which  determines  the  direction 
and  intensity  of  the  osmotic  movement  of  nutrients  from  the 
leaves  towards  the  respiring  cells.  Imbibed  water,  that  is  water 
from  an  external  source,  which  contains  no  nutrients  that  are 
directly  available  for  supporting  respiration,  tends  to  withdraw 
nutrients  from  any  cell  with  which  it  is  in  contact,  whereas  meta-  I 
bolic  water,  produced  by  the  oxidation  or  dehydration  of  organic  i 
nutrients  within  the  cell  walls,  tends  in  the  opposite  direction  ] 
and  attracts  nutrients  to  the  points  where  they  are  most  needed. 
The  first  leads  to  the  starvation  and  death  of  a cell,  the  second  to 
its  development  and  growth. 

The  respiring  cells  of  a mature  plant  are  nourished  in  the  ! 
same  way  as  those  in  the  sprouts  of  a germinating  seed,  before 
photosynthesis  is  established.  In  both  cases,  nutrients  are  ■ 
brought  to  the  cells  by  osmosis,  where  they  are  oxidized  and  i 
dehydrated  by  respiration,  in  the  same  manner,  giving  rise  to  : 
products  of  the  same  general  nature.  The  seedling,  however, 


Role  of  Metabolic  Water  in  Vital  Phenomena  159 

at  this  stage  of  development,  depends  for  its  growth  solely  npon 
a limited  store  of  nutrients  that  is  being  constantly  depleted, 
since  it  is  incapable  of  acquiring  organic  nutrients  from  an  ex- 
ternal source,  while  the  products  of  respiration  are  all  wasted. 
Photosynthesis  changes  this  condition,  each  leaf  becoming  a 
laboratory  in  which  carbon  dioxide  from  external  sources  to- 
gether with  a large  part  of  the  carbon  dioxide  resulting  from 
respiration,  and  practically  all  other  excreted  products  from  cells 
in  all  parts  of  the  plant,  are  elaborated  into  available  food  mate- 
rials. From  this  time  on,  new  sources  of  supply  distributed 
over  the  plant  add  to,  and  finally  wholly  replace  the  reserve 
stored  in  the  seed.  There  is  thus  provided  a surplus,  and  the 
distance  through  which  nutrients  must  be  carried  to  meet  the 
demands  of  respiring  cells,  is  greatly  diminished.  In  this  way 
energy  is  conserved  to  a plant  and  its  rate  of  growth  greatly 
augmented. 

i 

Water  Produced  in  Animal  Metabolism 

The  energy  required  for  maintaining  the  vital  functions  of 
animal  cells  is  derived  from  the  oxidation  of  nutrients,  through 
the  respiration  of  protoplasm,  in  a manner  entirely  analogous  to 
that  which  occurs  in  vegetable  cells.  In  both  cases,  the  nutrients 
are  .oxidized  within  the  cell  wall,  where  all  of  the  resulting  pro- 
ducts are  set  free.  These  products,  which  consist  principally  of 
carbon  dioxide  and  water,  are  gradually  removed  from  the  cell 
by  osmosis  and  diffusion.  In  consequence  of  these  changes,  there 
is  in  every  living  cell,  whether  it  be  vegetable  or  animal,  a con- 
tinual replacement  of  a part  of  the  organic  nutrients  by  water 
thus  maintaining  the  nutrient  solution,  within  the  cell  wall,  at 
a lower  concentration  than  in  the  fluids  which  distribute  the 
nutrients  to  the  tissues.  This  difference  in  concentration  insures 
a constant  movement  of  nutrients,  by  osmosis,  towards  the  points 
where  they  are  needed,  thus  providing  for  the  growth  and  main- 
tenance of  tissues,  so  long  as  suitable  nutrients  are  available  for 
the  purpose. 

In  this  respect,  no  difference  is  apparent  between  the  vegetable 
and  animal  kingdoms.  The  rate  of  oxidation  is,  however,  neces- 
sarily far  more  rapid  with  animals  than  with  plants,  in  order 
that  sufficient  energy  may  be  provided  for  maintaining  muscular 
activity.  This  demands  a more  abundant  supply  of  nutrients, 


1 60 


Wisconsin  Research  Bulletin  No.  22 


and  results  in  the  production  of  a larger  quantity  of  metabolic 
waste  materials,  per  unit  of  weight,  with  animals  than  with 
plants. 

Another  and  more  important  difference  is  the  inability  of 
animals  to  resynthesize  the  organic  waste  products  of  respiration 
into  substances  that  may  he  again  utilized  as  nutrients.  Most 
metabolic  products,  except  water,  if  allowed  to  accumulate,  exert 
a toxic  action  upon  animal  cells,  and  must  therefore  be  elimina- 
ted from  the  organism  as  fast  as  they  are  formed.  This  is  espe- 
cially the  case  with  the  soluble  products  arising  from  protein 
metabolism.  With  most  animals,  these  nitrogenous  products 
are  excreted  in  solution  through  the  kidneys,  chiefly  as  urea, 
but  birds,  reptiles,  and  all  insects  excrete  most  of  the  nitrogen- 
ous waste  matter  as  uric  acid,  or  its  ammonia  salt,  which  being 
practically  insoluble  in  the  body  fluids,  is  voided  in  a solid  con- 
dition. 

The  organic  nutrients  utilized  by  animals  are  all  derived  from 
external  sources  and  no  water  need  be  consumed  for  their  synthe- 
sis, as  is  the  case  with  plants  ; for  this  reason,  all  of  the  meta- 
bolic water  arising  from  the  oxidation  of  nutrients  by  an  animal, 
is  available  for  other  vital  processes,  and  for  replacing  evapora- 
ted and  excreted  water.  Moreover,  under  similar  atmospheric 
conditions,  the  loss  of  water  by  evaporation  is  far  greater  per 
unit  of  weight  with  plants  than  with  animals  because  of  the 
enormous  leaf  surface  exposed  directly  to  currents  of  air,  and 
because  in  the  case  of  animals  the  surface  is  usually  protected 
from  a rapid  change  in  hygroscopic  conditions  by  a covering  of 
hair,  wool,  or  feathers,  and  in  the  case  of  some  insects  in  the 
larval  state,  by  a silky  envelope.  For  these  reasons,  a much 
larger  proportion  of  the  water  required  for  the  vital  functions 
of  animals  is  supplied  by  metabolic  changes  in  the  food  and  tis- 
sues than  is  the  case  with  plants.  With  many  animals,  nearly 
all  of  the  water  required  is  derived  from  the  oxidation  of  organic 
nutrients  and  there  are  good  reasons  for  believing  that  meta- 
bolic water  would  he  sufficient  for  all  animal  needs,  were  it  not 
for  the  elimination  of  poisonous  nitrogenous  products  formed 
in  protein  metabolism. 

The  cell  walls  of  animal  tissues  are  mostly  protein  in  character 
while  those  of  vegetable  tissue  are  composed  of  carbohydrates. 
This  difference  in  composition  necessitates  a much  larger. pro* 


Role  of  Metabolic  Water  in  Vital  Phenomena  161 


portion  of  nitrogen  in  the  food  of  animals  than  in  that  of  plants, 
and  also  makes  the  poisonous  products  of  metabolism,  which  must 
be  removed,  very  large,  since  animals  are  provided  with  no 
means  for  regenerating  such  products  into  nutrients,  as  is 
brought  about  in  plants  by  photosynthesis. 

The  need  for  water  is  much  less  for  animals  that  excrete  uric 
acid  than  for  those  that  excrete  urea,  since  uric  aid,  being  prac- 
tically insoluble  in  the  body  fluids,  is  not  so  poisonous  as  urea 
and  is  voided  solid  with  a minimum  loss  of  water.  Many  ani- 
mals that  excrete  uric  acid,  instead  of  urea  never  have  access  to 
water  and  subsist  in  every  stage  of  their  development  upon 
air  dried  food  which  usually  contains  less  than  10  per  cent  water. 
The  most  striking  illustrations  of  this  kind  are  found  among  in- 
sects such  as  the  clothes  moths,  the  grain  weevils,  the  dry  wood 
borers,  the  bee  moths,  etc.  The  larvae  of  these  insects  contain 
a high  per  cent  of  water,  and  the  mature  forms,  in  spite  of  the 
development  of  wings  which  are  relatively  dry,  rarely  contain 
less  than  50  per  cent  water.  It  is  fair  to  assume  that  all  of  this 
water  is  Metabolic,  since  the  insects  never  have  direct  access  to 
water  and  it  is  extremely  doubtful  if  the  free  water  in  their 
food  is  sufficient  to  replace  the  water  lost  through  respiration, 
by  evaporation  from  the  surface,  and  as  a part  of  the  excreta. 

It  has  been  suggested  that  the  tissues  of  these  insects  are  hy- 
groscopic and  absorb  sufficient  water  from  the  surrounding  air 
to  supply  all  of  their  needs,  but  when  killed  the  bodies  of  these 
insects  dry  quickly  upon  exposure  to  ordinary  air,  a change 
that  could  not  occur  if  the  substances  that  compose  their  tissues 
were  inherently  hygroscopic.  In  this  respect  the  bodies  of  such 
insects  do  not  appear  to  differ  materially  from  other  animal 
tissues.  The  only  remaining  source  of  water  is  the  metabolic 
changes  in  the  food  and  tissues  which  appears  to  be  amply  suf- 
ficient for  all  purposes,  if  enough  suitable  food  is  available. 

For  several  years  observations  have  been  made  upon  insects, 
especially  those  capable  of  subsisting  upon  air-dry  materials, 
to  determine  the  minimum  water  requirement.  The  most  im- 
portant of  these  observations  are  here  given  in  detail. 

THE  CLOTHES  MOTH 

( Tinea  Pelliond! a Linn.) 

In  1905  several  clothes  moths  were  placed  in  a desiccator 
• the  air  of  which  was  dried  with  sulphuric  acid.  There  were 


162 


Wisconsin  Research  Bulletin  No.  22 


placed  in  the  same  vessel  pieces  of  woollen  cloth  that  had  been 
previously  dried  at  a temperature  approximating  97°  C.  The 
millers  remained  active  for  several  days,  none  dying  until  the 
tenth  day,  but  all  were  dead  by  the  fourteenth  day.  The  ma- 
ture insects  subsist  entirely  upon  nutrients  consumed  and  stored 
in  the  larval  state,  and  since  they  are  quite  active,  live  but  a 
short  time  under  the  most  favorable  conditions.  It  is  doubtful 
if  their  life  was  materially  curtailed  by  exposure  to  dry  air. 

Before  death,  these  millers  deposited  many  eggs  upon  the  dry 
cloth,  most  of  which  hatched,  the  young  larvae  being  seen  crawl- 
ing over  the  cloth.  The  larvae  varied  in  size,  and  particles  of 
excrement  were  found  in  the  vessel  indicating  that  the  larvae 
had  consumed  and  assimilated  some  of  the  dry  food.  No  live 
larvae  were  found  after  the  twentieth  day  following  the  confine- 
ment of  the  millers.  It  is  not  known  how  long  any  of  the  larvae 
lived,  under  these  conditions;  it  is  certain,  however,  that  the 
millers  laid  eggs  which  hatched,  and  that  the  young  larvae  lived 
several  days,  upon  dry  food,  in  an  atmosphere  practically  free 
from  moisture.  When  the  desiccator  was  opened,  at  the  end  of 
the  experiment,  the  confined  air  had  a distinct  odor  of  sulphur 
dioxide  and  it  was  thought  that  this,  rather  than  the  extreme 
dryness,  might  have  been  the  cause  of  death.  To  determine  this, 
a piece  of  dry  woolen  cloth  was  placed  in  a cage  containing  moth 
millers  and  left  there  several  days,  until  eggs  were  deposited 
upon  it ; when  these  eggs  began  to  hatch,  the  cloth  was  removed 
to  a ventilated  desiccator  containing  calcium  chloride.  Live 
larvae  were  seen  upon  the  cloth  every  day  for  two  weeks,  but 
none  were  discovered  after  this  time.  On  the  nineteenth  day 
the  cloth  was  removed  and  no  live  larvae  found. 

It  appears  from  these  experiments,  that  young  larvae  of  this 
species  of  insect,  cannot  long  survive  the  strain  induced  by  ex- 
posure to  dry  air.  It  has  been  suggested  that  the  larvae  in  the 
tests  lived  until  the  first  molting  period,  at  which  time  all  in- 
sects are  known  to  be  susceptible  to  adverse  conditions  of  any 
kind.  This  is  uncertain,  since  the  length  of  time  which  any  one 
larva  lived  is  unknown.  Be  that  as  it  may,  it  seems  far  more 
probable  that  excessive  evaporation  from  the  small,  thin  skinned 
larvae,  rather  than  the  dry  condition  of  the  food  provided,  was 
the  direct  cause  of  death,  in  these  cases.  The  rapid  develop- 
ment of  the  larvae,  in  the  open  air,  while  receiving  no  food  con- 
taining more  than  5 per  cent  water,  supports  this  view.  I have 


Bole  op  Metabolic  Water  in  Vital  Phenomena  163 


no  doubt  that  these  larvae  would  grow  to  maturity,  and  com- 
plete their  life  cycle  an  indefinite  number  of  times,  while  receiv- 
ing dry  food,  if  this  could  be  supplied  under  average  atmospher- 
ic conditions.  The  hygroscopic  nature  of  hair,  wool,  etc,  which 
constitute  the  natural  food  of  these  insects,  makes  an  experiment 
of  this  kind  impracticable. 

, At  another  time,  a mink  fur  collar  slightly  infested  with 
moths,  in  the  larval  stage,  was  placed  in  a breeding  cage  for  the 
insects  to  develop.  The  fur  was  in  a paper  sack  and  was  not 
removed  when  it  was  placed  in  the  cage,  although  the  sack  was 
left  open  to  allow  the  moths  to  fly  around  when  they  emerged 
from  the  cocoons.  A sample  of  the  fur  was  removed  for  a water 
determination,  the  result  of  which  is  given  later.  But  few  mil- 
lers appeared  from  the  first  brood  and  these  were  allowed  to  lay 
eggs  without  being  disturbed.  Three  months  after  the  fur  was 
put  in  the  cage,  it  contained  many  larvae  that  a month  later 
appeared  to  be  full  grown.  The  mature  larvae  were  active. 
Some  of  these  were  removed  for  analysis,  others  were  transfer- 
red to  another  cage  containing  wool  feathers,  a piece  of  astra- 
khan fur,  and  woolen  cloth,  the  water  contents  of  which  were 
known. 

Some  time  after  larvae  ceased  to  crawl  around,  in  the  first 
cage,  it  was  opened  and  carefully  examined  but  no  live  larvae 
were  found.  It  was  then  discovered  that  every  particle  of  fur 
had  been  consumed,  leaving  the  skin  clean  and  white.  The  silk 
trimmings  had  not  been  touched.  It  seems  probable  that  the 
larvae  left  in  the  cage  were  starved  to  death,  and  that  the  active 
movements  observed  were  made  in  search  of  food  and  not  to  find 
a secure  place  to  spin  a cocoon,  as  was  at  first  supposed.  The 
larvae  that  were  transferred  to  a cage  containing  different  food 
materials  completed  their  life  cycle  and  passed  through  several 
generations  during  the  next  six  years,  but  the  number  of  insects 
observed  at  any  one  time  was  small  compared  to  those  grown 
upon  the  mink  fur.  In  the  last  cage  the  insects  preferred  the 
astrakhan  fur  to  other  food  materials  present;  next  to  this  the 
wool,  no  difference  being  observed  between  the  washed  and  un- 
washed samples.  The  feathers  were  scarcely  touched. 

Water  determinations  were  made  in  the  la,rvae  of  the  clothes 
moth  at  three  different  times;  the  results  are  given  in  Table 
XVIII,  together  with  the  water  content  of  the  air  dry  material 


164 


Wisconsin  Research  Bulletin  No.  22 


upon  which  the  larvae  fed  while  passing  through  this  stage  of 
development. 


TABLE  XVIII.  WATER  IN  MOTHS  ANI)  IN  THEIR  FOOD 
Water  content  of  larvae  of  clothes  moths,  and  of  materials  upon  which  they  feed 


Water  Content  oe 

Larvae 

Water  Content  of  Food  Matertae 

Per  cent 

1 

Per  cent 

Size 

water 

. I 

Material 

water 

Full  grown 

57.66 

Woolen  cloth 

6.11 

Full  gl'own 

57.88 

Unwashed  wool 

7.56 

Medium 

59.83 

Washed  wool 

3.66 

Hair  from  fur 

9.08 

The  great  difference  in  water  content  between  the  food  ma- 
terials and  the  larvae  leaves  little  doubt  that  they  depend  chiefly 
upon  metabolic  water  for  all  of  their  vital  processes.  It  is 
doubtful  if  the  water  in  the  food  is,  in  any  case  sufficient  to 
replace  that  lost  in  normal  respiration.  Evaporation  from  the 
skin  is  small  in  any  case  and  in  the  mature  larvae  it  is  greatly 
reduced  by  an  envelope  consisting  of  particles  of  the  food  mate- 
rial bound  together  by  a silken  web.  It  seems  likely  that  the 
fur  supplied  more  favorable  conditions  for  development  than  did 
the  other  food  materials,  because  its  water  content  was  higher, 
and  that  this  is  the  reason  why  more  larvae  appeared  in  this 
than  in  wool.  There  were,  however,  some  individuals  that  passed 
through  both  the  larval  and  pupal  stages  in  all  of  the  materials. 
It  is  interesting  to  note  that  the  smaller,  growing  larvae,  in  which 
the  most  active  metabolism  occurred,  contained  the  highest  pro- 
portion of  water. 

Much  excrement  from  the  larvae  was  found  in  the  paper  sack 
enclosing  the  mink  fur  collar.  This  was  examined  by  Prof.  E. 
Y.  McCollum  and  a large  part  of  its  nitrogenous  constituents 
determined.  Of  the  total  weight  of  the  dry  samples  26.66  per 
cent  was  nitrogen;  10.22  per  cent  of  the  nitrogen  was  voided  as 
ammonia,  47.29  per  cent  as  uric  acid,  17.57  per  cent  as  urea, 
10  per  cent  as  creatin  and  creatinin,  .51  per  cent  as  purine  ba.ses, 
and  11.4  per  cent  was  insoluble  in  H Cl  and  NaOH. 

It  is  significant  that  so  large  a portion  of  the  excreted  nitro- 
gen consists  of  uric  acid,  which  contains  less  hydrogen  than  any 
other  known  nitrogen  compound  excreted  by  animals.  In  so  far 
as  this  has  occurred,  the  insects  have  derived  the  highest  possi- 


Role  of  Metabolic  Water  in  Vital  Phenomena 


165 


ble  amount  of  water  from  the  oxidation  of  nutrients.  -It  is  also 
important  that  the  amount  of  ammonia  in  the  excrement  has 
nearly  the  right  relation  to  uric  acid  to  form  ammonium  urate, 
a very  insoluble  compound  which  is  voided  solid  with  a mini- 
mum loss  of  water,  and  further,  on  account  of  the  insolubility 
of  ammonium  urate,  the  toxic  effects  of  both  ammonia  and  uric 
acid  are  practically  eliminated  so  that  little  water  is  wasted  in 
their  removal. 

These  insects  are  interesting,  not  only  because  they  subsist 
entirely  on  air  dried  food  containing  little  water,  but  especially 
because  their  food  materials  are  wholly  protein,  consisting 
chiefly  of  keratin,  a substance  that  is  not  attacked  by  the  diges- 
tive enzymes  of  any  other  class  of  animals  and  is  among  the 
most  resistant  of  animal  products  to  decay.  The  analysis  of  the 
excrement  shows  that  over  85  per  cent  of  the  excreted  nitrogen 
is  in  a different  form  from  that  in  the  food  material.  Since 
these  food  materials  are  wholly  protein  in  character,  it  is  cer- 
tain that  the  digestibility  of  hair  or  wool,  by  these,  insects,  is  at 
least  85  per  cent  of  the  amount  consumed.  The  digestibility 
undoubtedly  exceeds  this  because  it  is  unlikely  that  all  of  the 
metabolized  nitrogenous  products  were  identified.  It  is  note- 
worthy that  the  digestibility  of  keratin  by  these  insects  consi- 
derably exceeds  the  average  digestibility  of  vegetable  proteins 
by  the  higher  animals. 

Attempts  have  been  made  to  extract  from  the  moth  larvae  an 
enzyme  capable  of  dissolving  hair  and  wool  but  thus  far  with- 
out success.  Only  a few  larvae  were  available  for  this  purpose 
and  further  efforts  will  be  made  if  a sufficient  number  of  larvae 
can  be  obtained  at  one  time. 

the  bee  moth 
(Galleria  mellonella  Linn) 

The  larvae  of  these  insects  subsist  upon  empty  honey  comb, 
which  they  perforate  in  all  directions  by  tunnels  and  cover  with 
a fine  web  which  protects  them  from  the  attacks  of  bees.  The  ma- 
ture larvae  are  about  % inch  long  and  % inch  in  diameter,  and 
are  very  active.  They  are  most  abundant  in  abandoned  hives  and 
prefer  the  old  dark  colored  comb  to  that  which  is  clean  and 
white.  They  will  not  eat  clean  melted  wax. 

A quantity  of  infested  comb,  sent  to  the  Station  by  Carl  H. 


166 


Wisconsin  Research  Bulletin  No.  22 
* 

Hanson,  of  Elk  Mound,  Wis.,  supplied  material  for  the  follow- 
ing tests.  The  comb  contained  many  larvae  1-4  to  3-4  inches 
long.  The  water  content  of  the  full  grown  larvae  averaging 
170  milligrams  was  found  to  be  57.30  per  cent  and  that  of 
medium  sized  larvae  averaging  114  milligrams  was  59.24  per 
cent,  indicating  the  more  active  metabolism  by  the  younger 
insects  that  were  growing  rapidly. 

Some  of  the  larvae  were  placed  in  a breeding  cage,  upon 
honey  comb  which  they  ate  rapidly  and  in  a short  time  went  in- 
to the  pupal  state.  The  moths  that  issued  from  the  cocoons 
deposited  eggs  on  the  comb  and  the  sides  of  the  cage  which 
hatched  in  a few  days.  The  larvae  grew  rapidly  and  in  a few 
weeks  passed  into  the  pupal  state.  The  honey  comb,  upon 
which  the  larvae  fed,  contained  1.85  per  cent  water,  and  0.95 
per  cent  nitrogen.  The  larvae  had  access  to  no  water  from  any 
other  source  and  must  have  derived  practically  all  of  the  water 
required  for  their  vital  processes  from  metabolic  changes  in  the 
food. 

A piece'  of  comb  upon  which  eggs  had  been  deposited  was 
transferred  from  the  cage  to  a desiccator,  the  air  of  which  was 
dried  by  calcium  chloride.  They  hatched  as  soon  as  those  in  the 
open  air,  large  numbers  of  the  larvae  being  seen  crawling  over 
the  comb  and  the  sides  of  the  vessel  after  the  fourth  day.  This 
condition  continued  until  the  nineteenth  day,  when  no  more  live 
larvae  could  be  seen.  When  the  desiccator  was  opened  on  the 
twenty-third,  twenty-eighth,  and  thirty-sixth  days,  a number  of 
live  larvae  were  found  secreted  in  the  comb.  These  larvae 
were  neither  so  active  nor  so  large  as  those  grown  in  the  open 
air,  but  they  were  larger  .than  when  hatched.  The  excrement 
found  in  the  desiccator  supplies  additional  evidence  that  these  in- 
sects are  able  to  assimilate  dry  food,  in  practically  a dry  at- 
mosphere. Moreover  they  do  this  when  first  hatched,  during  a 
period  of  development  when  they  are  least  able  to  withstand 
adverse  conditions.  It  seems  probable  that  these  larvae,  as  well 
as  those  of  the  clothes  moths,  died  at  the  first  molting  period. 

Another  piece  of  comb  containing  a number  of  larvae  in 
different  stages  of  development  was  placed  in  the  desiccator. 
These  larvae  continued  to  grow;  some  of  them  spun  cocoons, 
and  27  days  after  the  comb  was  placed  in  the  desiccator  a moth 
appeared;  another  appeared  on  the  ninety-third  day  and  still 


Role  of  Metabolic  Water  in  Vital  Phenomena  167 

another  on  the  one  hundred  twelfth  day.  During  the  next  six 
weeks,  six  additional  moths  appeared  in  the  desiccator  and  at 
this  time  live  larvae  were  still  present  in  the  comb,  and  were 
growing.  Since  only  four  larvae  were  known  to  have  been  placed 
in  the  desiccator,  at  first,  it  is  certain  that  some  very  small  speci- 
mens escaped  observation.  It  is  possible  that  a few  eggs  in- 
troduced with  the  comb  may  have  hatched  and  passed  through 
every  stage  of  development.  It  is  certain  that  larvae  which 
have  passed  the  first  molting  period  are  capable  of  withstanding 
the  increased  evaporation  induced  by  dry  air,  even  at  subsequent 
molting  periods.  At  any  rate  they  are  far  more  resistant  than 
when  first  hatched.  In  this  case,  practically  all  the  water  re- 
quirements must  have  been  supplied  through  metabolic  changes 
in  the  dry  food,  since  the  comb  must  have  lost  nearly  all  of  its 
hygroscopic  water  in  the  desiccator. 

Beeswax,  the  chief  food  of  these  insects  is  resistant  to  ordi- 
nary solvents.  It  is  not  noticeably  hygroscopic,  but  it  contains 
a high  per  cent  of  hydrogen  and  when  assimilated  and  oxidized 
in  respiration,  it  yields  more  than  its  weight  of  water.  The  ni- 
trogen found  in  the  comb,  from  which  the  insects  derive  all  of 
their  protein  tissue,  is  probably  due  to  adhering  pollen  grains. 
Nitrogen  determinations,  by  Professor  E.  V.  McCollum,  in  the 
honey  comb  used  for  these  experiments  and  also  in  the  full 
grown,  live  larvae,  showed  the  nitrogen  content  of  honey  comb 
to  be  0.95  per  cent,  and  the  nitrogen  content  of  live  larvae  of 
the  bee  moth,  2.52  per  cent. 

Although  the  protein  content  of  the  food  consumed  by  the 
larvae  is  low,  the  bodies  of  the  insects  contain  fully  as  much 
nitrogen  as  is  found  in  other  classes  of  animals  which  feed  upon 
a far  narrower  ration. 

THE  FOUR-SPOTTED  PEA  WEEVIL 

( Bruckus  quadri-maculatus  Fabr.) 

Specimens  were  found  in  a package  of  seed  beans.  These 
were  transferred  to  a ventilated  cage  containing  sound  beans, 
and  in  about  three  months  mature  insects  appeared,  nearly 
every  bean  containing  two  or  more  perforations  made  by  the 
larvae.  It  was  impracticable  to  separate  a sufficient  number  of 
small  larvae  from  the  beans  for  a reliable  water  determination. 
Three  determinations  in  the  adult  weevils  gave  48.92,  51.26,  and 


1 68 


Wisconsin  Research  Bulletin  No.  22 


47.78  per  cent.  The  water  content  of  the  larvae  is  undoubtedly 
several  per  cent  higher  than  this.  The  beans  upon  which  these 
fed  exclusively  contained  8.38  per  cent  water. 

THE  CONFUSED  FLOUR  BEETLE 

( Tribolium  confusum  Duv.) 

These  were  found  in  a package  of  ground  red  pepper.  They 
were  transferred  to  other  vessels  containing  wheat  bran  and 
wheat  flour,  where  they  multiplied.  In  a few  weeks  insects  in 
every  stage  of  development  were  found  in  each  of  the  vessels. 
The  water  content  of  the  larvae  was  62.43  per  cent,  and  of  the 
mature  beetles  50.38  per  cent.  Water  was  not  determined  in 
the  food  materials,  but  it  is  not  likely  to  have  exceeded  10  per 
cent. 

Mature  insects  were  placed  in  a beaker  containing  wheat 
flour  that  had  been  dried  in  a steam  oven,  and  the  beaker  put 
into  a desiccator  over  sulphuric  acid.  The  beetles  burrowed  con- 
tinually in  the  dry  flour  and  probably  laid  eggs  although  none 
were  seen  and  no  larvae  were  found  in  the  flour.  No  dead 
beetles  were  seen  until  the  twentieth  day,  the  number  increasing 
from  this  time,  but  live  beetles  were  observed  until  the  fifty- 
eighth  day.  It  is  quite  certain  that  the  beetles  ate  some  of  the 
dry  flour,  and  that  their  life  was  prolonged,  thereby,  since  beetles 
confined  in  a vessel  containing  no  food,  in  the  open  air  where 
conditions  were  much  more  favorable,  all  starved  to  death  in 
thirty-six  days. 

The  natural  life  of  the  mature  beetles  is  longer  than  most 
other  mature  insects  as  in  shown  by  experiments  made  to  deter- 
mine whether  they  could  subsist  upon  food  materials  contain- 
ing only  a single  protein.  The  rations  supplied  to  the  beetles,  in 
these  experiments,  consisted  of  one  part  of  either  zein  or  edestin 
and  ten  parts  of  starch,  to  which  was  added  a little  milk  ash. 
A mixture  of  the  two  proteins  was  also  used.  In  addition, 
the  above  rations  were  all  duplicated  with  a little  cinnamon  oil 
added  to  each.  The  mixtures  were  placed  in  100  c.  c.  flasks 
that  were  loosely  stoppered,  to  prevent  escape  and  to  admit  air. 
Ten  mature  beetles  were  placed  in  each  flask.  No  dead  beetles 
were  seen  in  any  of  the  flasks  during  the  first  six  months,  and 
they  were  not  all  dead  at  the  end  of  11  bo  months.  No  live 
beetles  were  seen  after  a year.  There  was  apparently  no  differ- 


Role  of  Metabolic  Water  in  Vital  Phenomena  169 


ence  in  the  effect  of  the  different  mixtures.  No  larvae  were 
seen  at  any  time  in  any  of  the  flasks.  It  is  not  known  whether 
eggs  were  deposited  or  not.  This  may  have  occurred  and 
escaped  observation,  or  possibly  the  eggs  may  have  hatched  and 
the  young  larvae  been  devoured  by  the  beetles.  In  any  case, 
the  failure  to  reproduce  is  certain  evidence  that  none  of  the 
rations  were  entirely  suited  to  the  needs  of  the  insects.  It  is 
therefore  probable  that,  under  proper  conditions,  the  normal 
life  of  the  insects  is  more  than  a year. 

MEDITERRANEAN  FLOUR  MOTH 

( Ephestia  kuehniella  Zeller.) 

Larvae  were  supplied  by  the  department  of  economic  entomo- 
logy of  the  Station,  to  which  they  were  sent  for  identification. 
The  full  grown  larvae  contained  64.22  per  cent  water.  The 
water  content  of  the  food  upon  which  the  larvae  fed  was  not 
determined,  but  it  is  well  known  that  these  insects  pass  through 
every  stage  of  development  upon  food  containing  less  than 
10  per  cent  water. 

A sample  of  wheat  flour  middlings,  that  originally  contained 
10.62  per  cent  water,  was  infested  with  mites  not  identified. 
They  could  not  be  separated  from  adhering  material.  The 
sample  had  been  kept  several  months,  in  a covered  tin  can,  in 
the  laboratory  where  the  air  is  quite  dry,  and  it  is  unlikely  that 
it  had  absorbed  water  since  the  first  determination  was  made. 
The  water  content  of  the  mixture  of  mature  insects,  larvae  and 
adhering  material  was  15.98  per  cent ; a portion  of  the  flour, 
from  which  the  insects  were  separated  by  sifting  contained 
9.88  per  cent  water.  The  increased  water  content  of  the  flour 
is  entirely  due  to  the  metabolic  water  produced  by  the  respir- 
ation of  the  insects. 

The  water  content  of  insect  larvae  that  feed  upon  succulent 
a<^od  is  naturally  higher  than  in  insects  that  eat  only  dry  food. 

such  cases  the  production  of  metabolic  water  is  shown  by 
a higher  proportion  of  water  in  the  larvae  than  in  the  green 
food  they  eat.  This  is  well  illustrated  by  water  determinations 
in  the  larvae  of  the  tobacco  horn  worm,  ( Phlegetliontius  quinque- 
maculata,  Haworth.)  The  water  content  of  full  grown  larvae 
was  85.65  per  cent,  of  half  grown  larvae,  88.34  per  cent  and 
of  the  tobacco  leaves  which  formed  their  food,  82.78  per  cent. 


170  Wisconsin  Research  Bulletin  No.  22 

Here  as  in  all  other  cases,  the  younger  larvae,  in  which  the 
metabolic  processes  are  most  active,  contain  the  higher  per  cent 
of  water. 

Similar  determinations  in  larvae  of  the  common  green  cab- 
bage worm,  ( Pieris  rapae,  Linn.),  showed  the  water  content  of 
full  grown  larvae  to  be  83.33  per  cent,  of  medium  sized  larvae, 
84.26  per  cent,  and  of  the  cabbage  leaves  which  formed  their 
food  81.37  per  cent.  Here  again  the  larger  larvae  contain  the 
least  water. 

Water  Requirements  of  Animals 

The  amount  of  free  water  required  by  animals  depends,  pri- 
marily, upon  the  species  of  animal,  since  this  determines,  with- 
in narrow  limits,  the  nature  of  its  food  and  the  ultimate  pro- 
ducts of  protein  metabolism.  Carbohydrates  and  fats,  con- 
tained in  food,  may  be  completely  oxidized  through  respira- 
tion, the  products  being  carbon  dioxide  and  water.  The  car- 
bon dioxide  is  readily  eliminated  as  a gas  while  the  resulting 
water  is  added  to  the  body  fluids  and  utilized  for  the  main- 
tenance of  the  vital  functions.  The  water  produced  in  this 
way  is  more  than  sufficient  to  replace  the  normal  loss  of  water 
through  respiration  and  surface  evaporation  and  since  no  pois- 
onous substances  are  formed  in  the  complete  metabolism  of 
carbohydrates  or  of  fats,  there  would  be  no  need  of  free  water 
for  maintenance,  beyond  the  small  amount  always  present  in 
air-dry  food,  were  it  not  for  the  accumulation  of  mineral  con- 
stituents derived  from  the  food  and  the  poisonous  nature  of 
the  end  products  of  protein  metabolism,  the  most  of  which 
must  be  removed  in  solution. 

Where  provision  is  made  for  the  removal  of  these  harmful 
waste  products,  in  a solid  form,  as  is  the  case  with  all  insects 
that  subsist  upon  air-dry  materials,  there  is  no  need  for  a free 
water  supply  and  there  are  good  reasons  for  believing  that 
animals  of  this  class  may  develop  and  complete  their  life  cycle 
while  receiving  no  food  except  that  which  is  perfectly  dry. 

On  the  other  hand,  when  the  end  products  of  protein  meta- 
bolism are  soluble  in  the  body  fluids,  the  amount  of  water  u- 
sulting  from  oxidation  of  organic  nutrients  is  far  too  small  to 
keep  the  solution  of  these  substances  in  the  body  tissues  belo  v 
a toxic  concentration.  The  danger  is  increased  with  animals 


Dole  of  Metabolic  Water  in  Vital  Phenomena  171 


yielding  milk  and  with  birds  laying  eggs,  since  these  products 
remove  large  amounts  of  water  directly  from  the  body  fluids. 
Their  production  also  demands  the  consumption  of  a consider- 
able excess  of  protein  above  the  amount  required  for  normal 
maintenance,  the  waste  products  of  which  must  also  he  re- 
moved. In  these  cases,  an  extra  amount  of  water  from  exter- 
nal sources,  proportional  to  the  yields  of  milk  or  eggs  must  :>e 
supplied. 

The  energj"  expended  in  work  may  he  wholly  derived  from 
the  oxidation  of  carbohydrates  and  fats,  as  was  first  shown  by 
experiments  of  Fick  and  Wislicenus  in  1865  and  later  in  ex- 
periments by  Voit,  Pettenkoffer  and  Park  and  still  later  in 
experiments  of  Argutinsky,  Zuntz,  and  others.  It  is  however, 
impracticable  to  supply,  in  food,  sufficient  carbohydrates  and 
fats  for  the  maintenance  of  an  animal  performing  heavy  work, 
without  giving  some  extra  protein,  since  the  ordinary  grains 
and  feeds  contain  considerable  protein.  This  surplus  of  pro- 
tein is  eliminated  from  the  body  as  urea  and  an  increase  in  the 
supply  of  water  must  he  provided  for  the  purpose. 

The  increased  oxidation  of  nutrients  during  work  results 
in  the  production  of  an  increased  amount  of  heat  which,  unless 
removed  will  raise  the  temperature  of  the  body  above  normal. 
Danger  from  this  source  is  obviated  by  an  increased  evaporation 
of  water  from  the  lungs  and  from  the  whole  surface  of  animals 
that  perspire  freely.  The  water  derived  from  the  oxidation  of 
nutrients  is  insufficient  to  replace  the  extra  evaporation  during 
work  and  more  water  from  an  external  source  must  be  supplied 
for  this  purpose  than  when  an  animal  is  at  rest. 

The  water  requirement  of  mature  animals  that  excrete  urea, 
when  at  rest,  depends  chiefly  upon  the  amount  of  digestible 
protein  consumed.  For  a strictly  maintenance  ration  only 
sufficient  protein  need  be  assimilated  to  replace  the  loss  of  ni- 
trogen from  the  tissues,  and  to  furnish  material  for  the  natural 
loss  of  hair  etc.,  if  enough  carbohydrates  and  fats  are  provided 
to  meet  the  energy  and  heat  requirements.  Lnder  these  con- 
ditions, in  which  the  urea  excreted  is  a minimum  and  the  meta- 
bolic water  liberated  in  the  tissues  is  a maximum,  the  supply  of 
water  from  an  external  source  may  be  small. 

Serpents  and  other  reptiles  that  live  in  arid  regions  and 
rarely  if  ever  have  access  to  water,  except  that  contained  in 


172 


Wisconsin  Research  Bulletin  No.  22 


their  food  are  said  by  Vauquelin  to  excrete  all  of  the  waste 
nitrogen  as  salts  of  uric  acid.  The  same  is  true  of  birds  that 
live  on  desert  islands  where,  only  salt  water  is  available.  It 
is  essential  that  animals  of  these  types  should  produce  as  much 
metabolic  water  as  possible  from  the  assimilated  food,  and  the 
waste  of  water  through  the  excretions  should  be  reduced  to  a 
minimum.  Since  the  food  is  largely  protein  both  of  these  ends 
are  attained  by  the  excretion  of  uric  acid  which,  as  already 
stated,  contains  the  least  hydrogen  of  any  nitrogenous  sub- 
stance excreted  by  animals  so  that  the  maximum  amount  of 
metabolic  water  has  been  derived  from:  the  food  consumed. 
Brie  acid  and  its  salts,  however,  are  practically  insoluble  in 
vvcder  and  may  be  voided  in  a solid  condition  thereby  conserv- 
ing large  quantities  of  water  for  other  purposes  that  would  be 
lost  if  the  excreted  product  were  soluble  in  water  as  is  the  case 
with  urea,  the  usual  form  in  which  metabolized  nitrogen  is  ex- 
creted. 


Most  large  domestic  animals  excrete  the  metabolized  nitro- 
gen of  their  food  as  urea  which  is  soluble  in  the  body  fluids  and 
hence  requires  more  water  for  its  elimination  than  animals  that 
excrete  uric  acid  and  its  salts,  which  are  insoluble  and  usually 
voided  solid.  It  would  be  expected  also  that  animals  which 
excrete  urea  would  require  more  water  when  the  proportion  of 
protein  in  the  ration  is  increased.  Upon  this  point  Professor 
Henry1'  says,  “Possibly  due  to  their  laxative  nature,  feeds  rich 
m crude  protein  (bran,  linseed  meal,  peas,  etc.)  cause  a greater 
demand  for  water  than  starchy  feeds.”  And  again10  “On  pro- 
tein-rich feeds  the  pig  needs  more  water  than  when  on  starchy 
feeds.”  An  experiment  with  wide  and  narrow  rations  by 
Armsby20  shows  that  the  cows  fed  on  the  narrow  ration  drank 
more  water  (this  being  supplied  ad  libitum)  than  those  on  a wider 
ration.  There  were  two  sets  of  experiments  with  different  an- 
imals each  extending  over  three  weeks,  two  animals  being  fed 
in  each  case.  Table  XIX  shows  the  average  daily  amounts  of 
water  consumed  per  animal,  as  well  as  the  amount  of  water 
taken  per  pound  of  dry  matter  in  the  ration. 


18  Feeds  and  Feeding,  p.  63. 

10  Feeds  and  Feeding,  p.  563. 

20  Wis.  Expt.  Sta.  Rept.  1887.  p.  136, 


Role  of  Metabolic  Water  in  Vital  Phenomena  VI  o 


Table  XX,  compiled  from  results  obtained  by  Georgeson  of 
the  Kansas  Station  in  experiments  with  fattening  steers,  is 
copied  from  “ Feeds  and  Feeding  p.  327. 


TABLE  XIX.  INFLUENCE  OF  NUTRITIVE  RATIO  ON  WATER  INTAKE 


- — 

Water  Consumed 

Experiment 

Period 

Nutritive 

ratio 

Total  lbs. 
per  day 

Pounds  water 
per  lb. of 
dry  matter 

1 

Hi9 

7L1 

3.28 

•J  HQ 

2 

3 

1:11 

1:7.9 

D . up 

3.50 

II 

1 

1:6.1 

70.4 

74.7 

74.3 

3.24 

O AQ 

2 

3 

1 :4.9 
1:5.9 

3.40 

Many  other  experiments  might  be  cited  in  support  of  the 
hypothesis  that  animals  consume  more  water  when  the  protein 
in  the  ration  is  increased.  I believe  it  is  a general  principle, 
and  that  the  function  of  the  extra  water  is  to  facilitate  the  re- 
moval of  urea  from  the  system. 


TABLE  XX.  WATER  DRUNK  IN  WINTER  BY 


FATTENING  STEERS 


Water  drunk 


Lot 


Feed  given 


1 Corn  meal.  bran,  shorts,  oil  meal  with 

11.. ..  Corn  meal,  molasses,  and  corn  fodder. 

111..  Oil  cake,  hay 

IV. . . Ear  corn,  corn  fodder 


hay 


Lbs.  daily 

Lbs.  per  lb. 

per  steer 

of  feed 

79 

2.5 

73 

2.4 

91 

3.4 

56 

1.8 

Humans  and  other  large  animals  that  excrete  the  waste  nitro- 
gen products  of  protein  metabolism  chiefly  in  the  form  of  urea 
live  only  a short  time  if  deprived  of  water,  even  when  organic 
nutrients  are  supplied  in  abundance.  They  live  much  longei 
and  suffer  less  when  organic  nutrients  are  withheld  and  water 
is  supplied.  This  indicates  that  death,  in  the  first  instance,  is 
not  due  to  starvation  since  the  necessary  energy  for  maintain- 
ing vital  functions  is  provided ; the  metabolic  water  is  also 
more  than  sufficient  to  replace  the  water  lost  by  evaporation  and 
hv  respiration.  The  fact  that  an  abundant  supply  of  water  pro- 


174 


Wisconsin  Research  Bulletin  No.  22 


f!10Ufh  U contributes  no  energy  to  the  system,  in- 
ates.  that  death  ensues  when  water  is  withheld,  because  of 
n accumulation  of  end  products  of  metabolism,  in  this  case 
urea  e te,  which  cannot  be  eliminated  by  the  small  surplus  of 
metabolic  water  available  for  this  purpose.  In  other  words, 
eath  results  from  uremic  poisoning  rather  than  from  starvation 
urther  evidence  in  this  same  line  is  supplied  by  reptiles 
some  species  of  insects,  and  birds  that  void  the  nitrogen  end 
products  as  salts  of  uric  acid,  in  a solid  form.  Many  of  these 
are  capable  of  going  without  food  or  water  for  long  periods 
and  are  apparently  none  the  worse  for  the  fast.  In  this  case 
he  metabolic  water,  together  with  that  contained  in  the  food 
sufficient  for  all  purposes,  since  water  required  for  removing 
waste  products  is  reduced  to  a minimum.  I once  Knew  of  a hen 
that  lived  for  six  weeks  without  a particle  of  food  or  a drop  -,t 
water  and  although  extremely  emaciated  and  weak  when  dis- 
covered, she  quickly  regained  her  strength  and  weight  and  was 
soon  apparently  as  well  as  ever. 

Hibernating  animals,  which  subsist  chiefly,  upon  stored  body 
fat  and  m which  protein  metabolism  is  reduced  to  a minimum 
go  for  several  months  without  food  or  water,  metabolic  water 
being  ample  for  all  their  needs.  It  should  be  borne  in  mind  in 
this  connection  that  the  oxidation  of  fats  during  destructive 
metabolism  results  in  the  production  of  a.  greater  weight  of 
water  than  the  original  weight  of  fats  oxidized;  also  that  res- 
piration being  at  an  extremely  low  ebb  in  this  state,  the  loss 
of  water  through  the  lungs  is  very  small.  The  low  temperature 
en  s to  reduce  evaporation  from  the  skin  to  a minimum  Un- 
der these  conditions  the  amount  of  metabolic  water  may  be 
ample  for  removing  injurious  waste  products.  Another  pos- 
sibility is  that  animals  in  this  state  may  be  less  sensitive  to 
uremic  poisoning  than  when  active. 

There  are  many  animals  that  are  able  to  go  long  periods  with- 
out having  access  to  water  except  that  contained  in  their  food 
in  which  water  usually  amounts  to  less  than  20  per  cent  of  total 
weight,  and  the  metabolic  water  derived  from  oxidation  of 
organic  nutrients.  A notable  example  of  this  is  the  prairie  dog 
which  thrives  in  semi-arid  regions.  These  small  animals  feed 
upon  the  the  native  herbage  which  for  months  at.  a time  is  as 
dry  as  hay.  It  has  been  surmised  that  the  burrows  in  which 


Role  op  Metabolic  Water  in  Vital  Phenomena 


175 


they  live  extend  to  underground  water  courses,  but  this  does 
not  seem  likely  since  in  many  of  these  regions  weils  must  be 
sunk  hundreds  of  feet  before  water  is  reached.  It  is  more  prob- 
able that  they  depend  chiefly  upon  metabolic  water.  They 
feed  mostly  at  night  when  the  temperature  is  low  and  during 
the  hottest  hours  of  the  day  remain  in  their  burrows  where  the 
air  is  more  nearly  saturated  with  moisture  and  evaporation  is 
relatively  small. 

Mice  will  live  for  months  upon  air  dried  grain  containing 
about  10  per  cent  of  moisture,  and  under  these  conditions  will 
give  birth  to  young. 

Sheep,  when  in  pasture,  or  when  a part  of  their  ration  con- 
sists of  succulent  feed,  will  live  and  grow  for  an  indefinite  pe- 
riod, without  access  to  water,  although  they  thrive  better  when 
water  is  available.  In  this  case  the  thick  covering  of  wool 
serves  to  diminish  evaporation  from  the  skin,  and  the  relatively 
dry  feces  also  tend  to  conserve  water  for  other  purposes. 

The  camel  is  able  to  carry  heavy  burdens  over  stretches  of 
desert  for  many  days  without  drinking.  Before  starting  on 
a journey  where  water  is  not  available,  the  drivers  are  parti- 
cular to  have  the  animals  in  good  condition  with  the  humps  well 
stored  with  fat.  At  the  end  of  a trip  the  animals  are  poor  and 
the  fat  has  mostly  disappeared  from  the  humps.  The  rations 
fed  on  these  trips  are  chiefly  carbohydrate  concentrates  consist- 
ing of  dried  fruits,  bread,  etc,  containing  little  protein  so  that 
tire  nitrogen  waste  products  from  the  food  are  small  while  the 
metabolic  water  is  large.  Evaporation  of  water  from  the  skin 
is  greatly  reduced  by  a thick  coat  , of  fine  hair  and  very  little 
water  is  discharged  with  the  feces,  which  are  quite  dry.  It 
has  been  supposed  that  sufficient  water  was  stored  in  the  camel’s 
stomach  before  starting  to  supply  all  demands  of  the  trip.  It 
seems  likely,  however  that  metabolic  water  derived  from  oxi- 
dation of  body  fats,  and  of  food  rich  in  carbohydrates  is  of  far 
greater  importance  to  the  animal  than  the  stored  water. 

An  application  of  these  principles  would  undoubtedly  serve 
to  prolong  life,  when  suitable  water  for  drinking  is  not  avail- 
able. In  such  cases,  the  food  should  consist  of  carbohydrates 
and  fats.  Proteins  should  not  be  used.  In  this  way  vital  en- 
ergy may  be  maintained  with  a minimum  production  of  urea 
and  a maximum  amount  of  metabolic  water,  thereby  greatly  re- 


276 


Wisconsin  Research  Bulletin  No.  22 


ducmg  the  danger  from  uremic  poisoning.  There  need  be  no 
fear  of  starvation  nor  of  permanent  disability  even  when  pro- 
tein is  entirely  absent  from  a ration  for  several  weeks  if  enough 
carbohydrate  nutrients  are  provided  to  supply  the  necessary 
energy  for  maintaining  the  normal  vital  activities,  and  sufficient 
water  is  allowed  for  the  removal  of  the  small  amount  of  urea 
arising  under  these  conditions  from  the  waste  of  body  tissues. 
The  water  required  for  preventing  uremic  poisoning  under 
these  conditions  is  small  and  if  the  relative  humidity  of  the 
surrounding  air  is  high  enough  to  prevent  rapid  evaporation 
of  water  from  the  body,  the  metabolic  water  arising  from  the 
oxidation  of  nutrients  may  be  ample  for  the  purpose. 

Summary 

No  life  is  possible  except  in  the  presence  of  water,  which  is 
always  the  most  abundant  constituent  of  active  cells. 

A portion  of  the  water  found  in  living  organisms  is  derived 
from  external  sources  either  as  free  water,  or  as  a normal  con- 
stituent of  the  organic  nutrients  consumed.  Another  portion 
is  produced  within  the  organism  through  metabolic  changes  in 
its  food  and  tissues,  as  a result  of  respiration. 

The  chief  functions  of  water  are  to  dissolve  and  transfer 
nutrients  in  solution  to  active  cells,  to  remove  the  waste  pro- 
ducts of  metabolism  from  these  cells,  and,  in  the  chlorophyl 
bearing  cells  of  plants,  to  supply  material  for  the  synthesis  of 
organic  nutrients. 

The  reactions  involved  in  the  conversion  of  nutrients  to  a 
soluble  form  adapted  to  the  needs  of  living  cells  are  all  hydro- 
lytic in  nature.  That  is,  they  consist  in  adding  the  elements  of 
water  to  the  molecular  structure  of  the  nutrient.  These  changes 
are  effected  by  the  action  of  specific  enzymes,  peculiar  to  each 
type  of  organism,  which  are  produced  by  respiring  cells.  The 
reactions  involved  are  not  directly  dependent  upon  vital  pro- 
cesses and  for  the  most  part  they  may  be  brought  about  by 
chemical  means  without  contact  with  living  tissues. 

The  reactions  associated  with  the  growth  of  tissue  are  all 
dehydrating  in  character.  They  are  effected  by  energy  set  free 
through  respiration.  With  few  exceptions  these  reactions  have 
not  yet  been  brought  about  in  the  absence  of  living  protoplasm. 

Respiration  may  be  direct  or  intramolecular.  It  is  always 


Role  of  Metabolic  Water  in  Vital  Phenomena  177 


manifested  by  an  evolution  of  carbon  dioxide,  the  production 
cf  water,  and  the  liberation  of  energy  as  heat.  Direct  respi- 
ration can  occur  only  in  the  presence  of  free  oxygen.  In  the 
absence  of  free  oxygen  intramolecular  respiration  is  effected 
and  energy  set  free  through  a rearrangement  of  the  atoms  of 
the  molecules  comprising  the  nutrients  or  the  tissues,  which 
results  in  the  production  of  substances  of  a lower  order  than 
those  originally  present  in  the  active  cells  among  which  carbon 
dioxide  and  water  predominate. 

The  products  of  direct  respiration  (carbon  dioxide  and  water) 
are  not  directly  toxic  to  the  cell.  But  if  carbon  dioxide  is  per- 
mitted to  accumulate  until  it  excludes  free  oxygen  from  the 
cells,  intramolecular  respiration  intervenes  and  in  addition  to 
carbon  dioxide  and  water,  other  products  are  formed  which  in- 
terfere with  the  normal  activity  of  protoplasm.  Unless  these 
substances  are  removed  as  fast  as  formed,  the  death  of  the  cell 
results  in  a short  time.  But  if  these  substances  are  continually 
removed  and  replaced  by  suitable  nutrients,  vital  processes  may 
be  carried  on  indefinitely  with  but  little  direct  respiration. 
Growth  never  occurs  except  when  direct  respiration  is  possible. 

The  immediate  effect  of  respiration  is  to  remove  by  oxidation 
and  dehydration  a portion  of  the  nutrients  dissolved  in  the  cell 
fluids,  replacing  them  in  part  by  water,  and  thus  to  reduce  the 
concentration  of  the  solution  of  nutrients  within  the  cell  wall 
below  that  in  the  surrounding  fluids.  In  consequence  of  this 
there  is  established  an  osmotic  movement  of  organic  nutrients 
towards  an  active  cell  and  of  water  in  the  opposite  direction 
which  is  maintained  so  long  as  suitable  nutrients  are  supplied 
and  respiration  is  continued.  It  is  this  partial  replacement  of 
nutrients  by  metabolic  water  that  determines  the  direction  of 
the  movement  and  insures  a constant  supply  of  nutrients  to  all 
respiring  cells. 

In  every  seed  there  are  a few  active  cells  which  continue  to 
respire  and  to  perform  all  vital  functions,  so  long  as  the  seed 
is  viable,  and  as  respiration  invariably  results  in  the  produc- 
tion of  water,  there  must  always  be  a small  amount  of  water  in 
every  live  seed  or  spore.  It  is  impracticable  to  remove  all  of 
the  water  from  a seed,  except  by  the  application  of  heat.  Seeds 
of  corn,  exposed  to  air  dried  by  sulphuric  acid,  for  a period  of 
nearly  four  years,  still  contain  considerable  water.  The  con- 


Wisconsin  Research  Bulletin  No.  22 


178 

tinual  loss  of  dry  matter,  and  evolution  of  carbon  dioxide  are 
ample  evidence  of  respiration  throughout  the  whole  period. 

intramolecular  respiration  is  incapable  of  maintaining  the 
vital  processes  in  seeds,  for  a long  period.  Direct  respiration 
is  therefore  essential  to  the  continued  life  and  to  the  germi- 
nation of  a seed.  For  this  reason,  seeds  intended  for  planting 
should  always  be  stored  under  conditions  which  permit  a rather 
i ree  circulation  of  air.  They  should  never  be  stored  for  a long 
time  in  bulk,  in  tight  bins.  The  water  content  of  seed  grains 
should  be  reduced  soon  after  harvest,  to  not  more  than  10  per 
cent,  since  the  rate  of  respiration  is  dependent  upon  their  water 
content. 

The  specific  enzymes  required  for  the  conversion  of  the  stored 
nutrients  of  a seed  into  available  forms  are  absent  in  the  im- 
mature seed,  and  only  appear  in  the  mature  seed  after  direct 
respiration  is  established;  the  rate  of  their  formation  is  pro- 
portional to  the  rate  of  direct  respiration. 

The  water  content  of  the  sprouts  of  germinating  seeds  is  al- 
ways much  higher  than  that  of  the  remainder  of  a seed ; this 
is  due  to  the  production  of  metabolic  water  by  the  rapid  res- 
piration of  the  young  cells  in  these  tissues,  and  to  the  fact  that 
no  respiration  occurs  in  that  portion  of  the  seed  in  which  the 
nutrients  are  stored. 

( arbohydrates  of  various  degrees' of  hydration  are  found  in 
all  plants;  the  highest  state  of  hydration  is  represented  by  dex- 
trose and  levulose,  the  lowest  by  cellulose  and  siarch,  and  be- 
tween these,  many  intermediate  forms  occur.  The  first,  stable 
carbohydrate  to  appear  in  plants  is  starch  which  is  formed 
initially  in  leaves  by  photosynthesis.  This  insoluble  starch  is 
hydrolized  in  the  leaves  by  a diastatic  enzyme  and  changed  into 
a soluble  carbohydrate',  usually  dextrose,  and  carried  by  osmosis 
to  active  cells  in  all  parts  of  the  plant.  Within  these  cells,  a 
portion  of  the  dextrose  is  completely  oxidized  to  carbon  dioxide 
and  water  and  through  the  energy  liberated  in  the  reaction 
other  portions  are  dehydrated  in  various  degrees  giving  rise  to 
cellulose,  starch,  cane  sugar,  etc.  The  resulting  cellulose  re- 
mains within  the  cell  to  form  the  cell  wall,  the  starch  is  deposited 
as  a reserve  nutrient  to  be  drawn  upon  when  food  from  other 
sources  fails,  the  soluble  waste  products  are  removed  from  the 
cell  by  osmosis  and  mostly  carried  back  to  the  leaves  by  the 


Role  of  Metabolic  Water  in  Vital  Phenomena  179 


sap  where  they  are  once  more  converted  into  suitable  nutrients 
and  distributed  again  to  the  active  cells.  This  cycle  may  be 
completed  an  indefinite  number  of  times  by  the  same  carbon 
nucleus  until  it  is  finally  deposited  as  permanent  tissue  or  is 
completely  oxidized  to  supply  energy  for  maintaining  vital 
functions.  At  every  round  water  is  transferred  in  organic 
combination  from  the  leaves  to  every  growing  cell.  Nearly  all 
of  the  water  in  the  growing  cells  of  a plant  is  brought  to  them, 
in  this  manner,  in  organic  combination,  and  liberated  within 
the  cell  walls  by  the  process  of  respiration.  The  prevailing 
movement  of  liquid  water  is  away  from  rather  than  toward 
these  centers. 

The  ’free  oxygen  required  for  respiration  during  the  germina- 
tion of  seeds  may  be  derived  from  a solution  of  hydrogen  per- 
oxide in  which  the  seeds  are  immersed.  This  method  of  testing 
germination  has  an  advantage  over  the  usual  practice  of  plac- 
ing the  seeds  between  wet  cloths,  since  the  growth  of  molds, 
mildews,  etc,  which  interfere  with  germination,  is  prevented. 

Dry  starch  combines  directly  with  water  in  a manner  analo- 
gous to  substances  which  crystallize  with  water  of  crystallization. 
This  molecular  combination  always  precedes  the  hydrolysis  of 
starch  to  dextrose  by  a diastatic  ferment. 

The  heat  generated  in  the  preliminary  combination  of  starch 
with  water  is  practically  two  thirds  of  the  theoretical  amount 
set  free  in  the  change  from  starch  to  dextrose.  No  change  in 
temperature  occurs  when  a boiled  starch  paste  is  hydrolized  by 
diastase  to  dextrose,  since  the  heat  absorbed  in  the  solution  of 
ihe  anhydrous  dextrose  formed  is  equal  to  that  set  free  when 
hydrated  starch  is  hydrolized. 

The  final  ripening  changes  in  most  fruits  proceed  fully  as 
rapidly  after  removal  from  the  tree  as  when  left  undisturbed. 
These  changes  are  the  result  of  direct  respiration  of  living  cells 
in  the  fruit  which  continue  to  function  after  the  fruit  is  picked. 
The  increase  in  succulence  during  ripening  is  partly  due  to  the 
production  of  metabolic  water  through  respiration  and  partly 
to  the  increased  solubility  of  the  products  formed.  It  is  not 
due  to  water  derived  from  the  parent  plant.  The  water  con- 
tent is  proportionately  greater  in  ripe  fruit  than  in  green  fruit, 
in  spite  of  considerable  loss  of  water  through  evaporation',  even 
though  the  fruit  be  ripened  off  the  tree. 


180  Wisconsin  Research  Bulletin  No.  22 

Fruits  do  not  ripen  normally  when  oxygen  is  excluded.  In 
this  case  the  changes  are  similar  to  those  that  occur  in  the  en- 
siling of  succulent  plant  tissues  of  any  kind.  They  are  usually 
associated  with  the  production  of  organic  acids  of  various  kinds. 

Water  absorbed  from  the  soil  through  the  roots  by  capillarity 
passes  quite  directly  through  the  tracheids  to  the  leaves,  picking 
up  on  the  way  by  solution,  carbon  dioxide  and  other  waste  pro- 
ducts of  cellular  respiration  from  all  of  the  living  tissues  of  the 
plant.  By  the  action  of  light,  all  of  these  waste  products  in  the 
leaves  are  reorganized  into  suitable  nutrients  and  again  dis- 
tributed from  cell  .to  cell  by  osmosis. 

The  continual  addition  of  carbon  dioxide  from  respiring  cells 
to  the  sap  as  it  passes  through  the  tracheids  towards  the  leaves 
and  the  varying  solubility  of  carbon  dioxide  in  the  sap  with 
change  of  temperature,  are  most  important  factors  in  develop- 
ing sap  pressure  and  maintaining  its  movement  at  all  periods  of 
growth. 

Through  photosynthesis  practically  all  of  the  waste  products 
of  plant  metabolism  are  converted  into  nutrient  substances  and 
used  again  for  maintaining  vital  processes.  In  this  way,  the 
loss  of  organic  nutrients  is  reduced  to  a minimum  and  the  rate 
of  plant  growth  greatly  augmented. 

Animal  cells  respire  in  a similar  manner  to  vegetable  cells 
and  with  the  same  general  effect.  The  oxidation  of  nutrients 
and  production  of  metabolic  water  within  the  cell  wall  constantly 
maintains  the  solution  of  nutrients  in  the  cell  fluids  at  a lower 
concentration  than  in  the  blood  which  distributes  nutrients  to 
the  tissues.  This  difference  in  concentration  insures  a constant 
movement  by  osmosis  of  soluble  nutrients  through  the  cell  wall 
to  replace  the  material  that  has  been  destroyed  by  respiration 
or  removed  from  solution  for  the  growth  of  tissue. 

The  most  important  difference  between  vegetable  and  animal 
metabolism  is  the  inability  of  animals  to  reconvert  the  waste 
products  of  .respiration  into  suitable  nutrients.  Most  of  these 
products  are  toxic  to  animal  cells  and  must  be  eliminated  from 
the  tissues  as  fast  as  formed.  This  is  especially  the  case  with 
the  waste  products  of  protein  metabolism.  Most  animals  excrete 
these  products  in  solution  through  the  kidneys  chiefly  as  urea, 
but  insects,  birds,  and  reptiles  excrete  insoluble  salts  of  uric 
acid  in  a solid  form  with  a minimum  loss  of  water.  It  is  sig- 


Role  of  Metabolic  Water  in  Vital  Phenomena  181 

nificant  in  this  connection  that  uric  acid  contains  the  least 
hydrogen  of  any  nitrogenous  compound  excreted  by  animals, 
thus  securing  for  the  animals  that  eliminate  their  nitrogenous 
waste  products  in  this  form  the  maximum  amount  of  metabolic 
water. 

Animals  that  excrete  urea  require  a liberal  supply  of  water, 
aside  from  that  normally  contained  in  the  food,  to  keep  the 
urea  content  of  the  blood  below  a toxic  concentration.  The 
water  requirements  of  these  animals  depend  largely  upon  the 
amount  of  protein  consumed.  On  a protein  free  diet  the  meta- 
bolic water  together  with  the  free  w^ater  contained  in  the  food 
is  sufficient  to  maintain  all  vital  processes,  to  remove  poisonous 
excretions,  and  to  replace  water  lost  in  respiration  and  in  eva- 
poration from  the  body  for  long  periods,  as  is  shown  by  hiber- 
nating animals  that  subsist  chiefly  upon  body  fat. 

Many  varieties  of  insects  and  other  animals  that  excrete  the 
waste  products  of  protein  metabolism  as  salts  of  uric  acid  in 
solid  form  require  no  free  water  at  any  time,  except  the  small 
amount  present  in  air  dried  food,  the  water  content  of  which 
is  usually  less  than  10  per  cent.  This  is  possible  because  the 
insoluble  nature  of  uric  acid  renders  it  but  sightly  poisonous 
and  permits  of  its  excretion  with  a minimum  loss  of  water. 
This  is  the  case  with  the  clothes  moths,  the  grain  weevils,  the 
bee  moth,  and  a large  number  of  insects  that  live  upon  air 
dried  food  throughout  every  stage  of  their  development.  The 
larvae  of  these  insects  contain  from  five  to  ten  times  the  amount 
of  free  water  contained  in  their  food.  Some  of  these  insects 
are  capable  of  living  long  periods  upon  dry  food  in  an  atmos- 
phere containing  no  moisture.  No  doubt  they  would  live  in- 
definitely upon  dry  food  if  this  could  be  supplied  without  ex- 
posure to  dry  air  which  enormously  increases  the  loss  of  water 
by  evaporation. 

Metabolic  water  derived  from  the  oxidation  of  organic  nutri- 
ents would  probably  be  sufficient  for  all  animal  needs  were  it 
not  for  the  elimination  of  poisonous  substances  resulting  from 
protein  degeneration. 


, 


■ 


I&MoaoL 


Relation  of  Soil  Bacteria  to  Evaporation 

CONRAD  HOFFMANN ‘ 


Introduction 

Numerous  articles  and  parts  of  many  textbooks  have  been 
written  dealing  entirely  with  the  water  supply  of  soils,  but  in  all 
cases  the  discussions  concerning  the  subject  have  been  confined 
largely  to  physical,  and  less  frequently  to  chemical  considera- 
tions. Mention  is  made  of  capillarity,  gravity,  viscosity,  surface 
tension,  evaporation,  hygroscopic  moisture,  etc.  The  laws  gov- 
erning these  factors  liaAe  been  very  largely  determined  and  are 
now  facts  known  to  many.  But  aside  from  the  consideration  of 
the  physical  and  chemical  agencies  affecting  soil  moisture,  there 
are  certain  biological  factors  in  the  soil  which  undoubtedly  are 
closely  connected  with  the  movements  of  soil  water.  Owing  to 
the  prevailing  tendency  of  the  earlier  soil  bacteriologists  to  con- 
fine their  efforts  largely  to  problems  concerned  with  the  nitro- 
gen supply  of  soils,  and  those  more  or  less  closely  allied  thereto, 
other  fields  of  profitable  investigation  have  been  somewhat  neg- 
lected. Thus,  practically  no  work  has  been  performed  to  inves- 
tigate what  relations,  if  any,  exist  between  the  movement  of  soil 
water  and  bacterial  activities  in  the  soil. 

Capillarity  is  to  a large  extent  dependent  upon  the  surface 
tension  of  the  soil  w^ater;  thus  the  upward  movement  of  water 
taking  place  as  a result  of  capillarity  in  soils  must  also  be  in- 
fluenced by  the  surface  tension.  It  is  accordingly  .reasonable  to 
suppose  that  evaporation  at  the  surface  must  therefore  be  in- 
fluenced. It  is  a well  known  fact  that  the  addition  of  certain 
fertilizers  to  soil  increases  the  surface  tension  of  the  soil  water. 
Soluble  organic  matter  even  in  minute  quantities,  particularly 

1 Much  of  the  experimental  work  herein  reported  was  performed  un- 
der direction  of  the  author,  by  J.  E.  Graul  and  A.  A.  Vass. 


184 


Wisconsin  Research  Bulletin  No.  23 


that  of  an  oily  nature,  greatly  reduces  the  surface  tension.  As 
bacteria  in  their  metabolism  undoubtedly  form  * considerable 
quantities  of  soluble  compounds  both  organic  and  inorganic,  it 
is  reasonable  to  suppose  that  the  soil  bacteria  can  and  do,  in- 
fluence the  rate  of  evaporation  from  soils,  as  well  as  the  move- 
ment of  soil  water. 

In  recognition  of  this  fact  the  work  herein  reported  was  under- 
taken to  determine,  if  any  exist,  the  true  relation  between  the 
soil  bacteria  and  the  rate  of  evaporation  of  moisture  from  the 
soil. 

Stigell2  reports  a few  preliminary  experiments  relative  to  the 
influence  of  soil  bacteria  upon  evaporation.  His  results  indi- 
cate that  the  growth  of  bacteria  exerts  a retarding  influence 
on  the  rate  of  evaporation.  He  attributes  this  retardation  to 
the  following  possible  factors:  The  absorption  and  subsequent 
retention  of  moisture  by  the  bacteria  themselves;  and  changes  in 
the  physical  and  chemical  properties  of  the  soil  brought  about 
by  the  metabolic  activities  of  the  bacteria. 

Briefly,  his  method  of  procedure  was  to  employ  large  Petri 
dishes  containing  a layer  of  quartz  sand,  moistened  with  bouillon 
cultures  of  various  organisms.  Weighings  were  made  from  day 
to  day  and  the  losses  calculated  as  due  to  evaporation.  Series 
of  sterile  dishes  were  similarly  prepared  to  serve  as  controls. 
In  this  way  he  found  that  the  presence  of  bacteria  apparently 
retarded  the  rate  of  evaporation. 

Similar  experiments  were  next  undertaken  by  the  writer  in 
Dr.  Alfred  Koch’s  laboratory  at  Gottingen.  Here  however, 
results  were  obtained  which  apparently  contradicted  those  of 
Stigell.  In  fact,  instead  of  a retardation  there  appeared  to  be 
an  acceleration  in  the  rate  of  evaporation. 

Development  of  Technique 

It  was  found  that  the  technique  recommended  by  Stigell  intro- 
duced a factor  of  error,  which  frequently  was  so  large  as  to 
cover  the  slight  differences  which  Stigell  had  obtained  between 
sterile  and  inoculated  plates.  In  order  to  secure  more  reliable 
results,  it  was  found  advisable  to  run  at  least  four  and  prefer- 
ably six  plates  to  a set,  instead  of  only  one,  using  the  average 


2 Centbl.  Bakt.  Abt.  II,  1908,  21,  p.  60. 


Relation  of  Soil  Bacteria  to  Evaporation 


185 


for  final  results.  Even  where  the  utmost  precautions  to  avoid 
errors  were  observed,  wide  variations  were  frequently  obtained 
in  the  individual  dishes  of  any  given  set. 

Such  factors  as  temperature  and  the  humidity  of  the  air 
could  not  be  controlled  under  ordinary  room  conditions.  These 
factors  would  of  course,  alter  the  rate  of  evaporation  from  day 
to  day,  being  more  rapid  on  a dry,  sunshiny  day  than  on  a rainy, 
dark  day.  To  avoid  fluctuations  due  to  air  currents,  proximity 
to  radiators,  etc.,  the  experiments  were  conducted  in  a small 
room  separated  from  the  rest  of  the  laboratory.  All  plates 
were  placed  under  uniform  conditions  at  equal  distances  from 
the  walls  of  the  room,  and  all  plates  in  any  given  series  were 
placed  at  the  same  level.  The  control  and  the  normal  plates  were 
alternated  on  the  shelves,  rather  than  placing  all  normals  or  all 
controls  adjacent  to  one  another.  In  this  way  it  was  possible 
to  expose  all  plates  (normal  and  control)  to  identical  conditions, 
and  any  fluctuations  occurring  would  influence  all  dishes  alike. 
Differences  in  the  rate  of  evaporation  between  sets  of  the  various 
series  would  then  be  attributed  to  differences  in  treatment  rather 
than  m exposure,  and  comparisons  could  thus  be  made  with  a 
greater  degree  of  accuracy. 

Several  experiments  were  conducted,  employing  dishes  rang- 
ing from  7 to  21  cm.  in  diameter,  the  soil  layer  varying  from  2 
to  10  cm.  deep.  While  it  was  found  that  results  were  some- 
what more  uniform  when  a comparatively  small  evaporating 
surface  and  a deep  soil  layer  were  employed,  the  rate  of  evapo- 
ration under  such  conditions  was  so  slow  as  to  render  their  em- 
ployment undesirable  for  the  work  in  hand.  The  larger  evaporat- 
ing surfaces  obviously  increased  the  rate  of  loss  due  to  evapora- 
tion, but  at  the  same  time  increased  the  error,  that  is,  the  maxi- 
mum variation  between  the  individual  dishes  of  any  one  set.  By 
exercising  care  in  the  preparation  of  the  plates  this  maximum 
variation  between  individual  dishes  was  reduced  to  less  than  2.5 
grams  where  six  plates  were  used  in  a set.  It  was  finally  decided 
to  use  dishes  19  to  22  'em.  in  diameter  holding  soil  or  sand  1.8  to 
2.5  cm.  deep.  In  any  one  experiment  all  dishes  employed  were 
of  the  same  dimensions. 

Part  of  the  preliminary  work  was  performed  with  quartz 
sand  as  a substratum,  but  far  more  uniform  results  were  se- 
cured with  ordinary  dried  and  sieved  soil  as  a substratum,  this 


186 


Wisconsin  Research  Bulletin  No.  23 


being  undoubtedly  due  to  the  fact  that  soil  offers  more  continu- 
ous passages  for  capillary  movements  of  the  soil  moisture. 
Where  sand  was  employed,  it  was  necessary  to  add  nutrient  solu- 
tions if  luxuriant  bacterial  development  was  desired.  This  ad- 
dition complicated  the  experiments,  and  produced  conditions  far 
from  those  normally  and  naturally  existing  in  the  field.  Be- 
cause of  these  facts  soil  was  used  instead  of  sand  as  a substra- 
tum, sand  being  used  occasionally  for  comparisons.  The  soil 
used  was  allowed  to  air-dry,  and  was  then  passed  through  a 
twelve-mesh  sieve.  This  was  used  throughout  all  experiments 
unless  otherwise  stated. 

To  measure  the  influence  of  bacterial  growth  upon  the  evapo- 
ration, it  was  necessary  to  run  duplicate  series  kept  under  sterile 
conditions.  As  the  plates  were  exposed  directly  to  the  air,  they 
could  not  be  protected  from  contamination  after  preparation. 
Owing  to  the  nature  of  the  experiment,  it  was  impossible  to 
cover  the  plates  in  any  way  to  avoid  this  outside  contamination. 
As  the  rate  of  evaporation  from  the  soil  is  influenced  by  the  ex- 
tent of  capillary  action,  and  as  capillarity  is  to  a large  extent  de- 
pendent upon  the  surface  tension  of  the  soil  water,  it  seemed 
inadvisable  to  add  any  disinfectants  to  the  series  to  be  kept 
sterile.  The  addition  of  any  such  soluble  substances  to  the 
sterile  series  and  not  to  the  normal,  where  of  course  they 
could  not  be  added  if  growth  were  desired,  would,  it  was 
thought,  alter  the  surface  tension  of  the  sterile  series  and  so  in- 
fluence directly  the  capillarity,  and  indirectly  the  rate  of  evapor- 
ation. Ordinary  heat  sterilization  would  not  suffice,  fo,r  upon 
exposure  to  the  air,  contamination  would  immediately  result. 

Several  experiments  were  conducted  with  a dilute  mercuric 
chloride  solution  as  a means  of  sterilizing  the  soils  of  one  set 
in  contrast  to  a duplicate  set  sterilized  by  heat.  Twelve  plates 
each  containing  600  grams  of  soil  were  sterilized  by  heating. 
To  each  of  six  were  then  added  200  c.  c.  of  a 1 to  1000  solution 
of  mercuric  chloride  made  with  distilled  water.  To  the  other 
six,  200  c.  c.  of  sterile  distilled  water  were  added.  All  plates 
were  kept  in  a drying  oven  to  avoid  so  far  as  possible  the  con- 
tamination of  the  set  not  treated  with  mercuric  chloride.  Thus 
all  were  identically  treated  except  that  mercuric  chloride  was 
added  to  one  set  of  six.  Daily  weighings  were  made  to  determine 


Delation  of  Soil  Bacteria  to  BVaporatIon  187 

the  losses  of  moisture  due  to  evaporation.  The  averages  of  the 
six  plates  in  each  set  are  given  in  Table  I. 

It  is  apparent  from  the  data  that  the  mercuric  chloride  ex- 
erted no  appreciable  influence  either  in  increasing  or  decreasing 
the  rate  of  evaporation,  as  was  at  first  expected.  This  being 
true,  a dilute  mercuric  chloride  solution  was  used  to  produce 
and  maintain  sterile  conditions  when  desired. 

TABLE  I.  INFLUENCE  OF  MERCURIC  CHLORIDE  SOLUTION  ON  RATE  OF 

EVAPORATION 


Loss  of  moisture  in  grams  for  the  periods 
indicated 


1st 

day 

2nd 

day 

3rd 

day 

4th 

day 

5th 

day 

6th 

day 

7th 

day 

To- 

tal 

HgClg  set  ( Av.  of  6 plates) 

12.2 

6.5 

8.8 

7.9 

7.2 

12.5 

14.3 

69.4 

H#0  set  (Av.  of  6 plates) 

12.4 

6.9 

8.3 

7.3 

7.0 

12.8 

13.7 

68.4 

Difference  in  favor  of  HgCl2  set 

—0.2 

—0.4 

+0.5 

+0.6 

+0.2 

—0.3 

+0.6 

+1.0 

The  technique  finally  adopted  as  a result  of  the  preliminary 
work  is  briefly  summarized  as  follows: 

Circular  dishes  eitherl9.1  or  21  c.  c.  in  diameter  were  employed. 
As  a substratum,  air-dried  soil  passed  through  a 12-mesh  sieve 
was  used  in  all  cases  unless  otherwise  stated.  For  each  plate 
500  or  600  grams  of  soil  were  used,  which  when  uniformly 
packed  and  leveled,  made  a soil  layer  2.8  cm.  deep  in  the  larger 
dishes  and  3.2  cm.  in  the  smaller  dishes.  To  each  of  the  plates 
were  then  added  by  means  of  a 50  c.  c.  pipette,  150  to  300  c.  c. 
of  distilled  water  depending  upon  the  nature  and  amount  of 
soil  employed,  the  water  being  applied  either  pure  or  modified 
as  indicated  under  the  individual  experiments.  It  was  planned 
to  have  approximately  30%  of  moisture  present  initially  in  all 
experiments,  thus  affording  ample  moisture  for  vigorous  bac- 
terial multiplication.  Great  care  was  exercised  to  have  the 
control  sets  differ  from  the  normals  in  only  the  one  respect  de- 
sired, so  that  any  differences  occurring  might  be  attributed  to 
the  one  particular  modification  in  treatment.  In  all  cases  the 
total  initial  weight  was  recorded  and  subsequent  weighings  were 
then  made  at  more  or  less  frequent  intervals  to  determine  the 
rate  of  evaporation.  All  weighings  were  made  upon  a torsion 


188 


Wisconsin  Research  Bulletin  No.  23 


balance  weighing  to  within  1/10  of  a gram.  The  results  of  the 
individual  dishes  of  each  set  were  then  added  and  the  average 
taken  to  indicate  the  loss  due  to  evaporation  occurring  from 
period  to  period. 

Problem  I.  Influence  of  Gelatin  on  Rate  of  Evaporation 

Theoretically  it  would  seem  plausible  to  expect  that  bacterial 
growth,  particularly  if  profuse,  would  tend  to  retard  evapora- 
tion, as  with  profuse  growth  a more  or  less  viscid  condition 
obtains.  The  effect  of  such  a condition  would  be  purely  physi- 
cal and  analogous  to  what  one  would  expect  from  the  addition 
of  a dilute  gelatin  solution  instead  of  distilled  water.  The 
moisture,  it  would  seem,  would  be  retained  by  the  gelatinous 
nature  of  the  bacterial  growth  in  the  first  case  and  of  gelatin 
itself  in  the  latter. 

Whether  gelatin  would  occasion  such  a retention  of  moisture 
thereby  preventing  to  a greater  or  less  degree  its  evaporation, 
was  thought  worthy  of  investigation  before  proceeding  further. 
Several  experiments  were  accordingly  performed  employing 
a gelatin  solution  (approximately  2%)  in  contrast  to  ordinary 
distilled  water,  both  under  sterile  conditions  and  in  normal  soil 
m which  bacteria  were  allowed  to  develop.  The  results  of  these 
experiments  are  recorded  in  Tables  II  and  III  and  represent 
the  averages  of  the  four  plates  used  for  each  set. 

It  would  appear  that  the  gelatin  solution  employed  had  but 
little  effect  upon  the  evaporation.  If  any  influence  was  exerted, 
it  was  at  most  a slight  retardation,  this  being  more  evident  in 
the  normal  soil  sets.  The  differences  however,  between  the  nor- 
mal distilled  water  and  the  dilute  gelatin  sets  fall  largely  within 
the  limits  of  the  factor  of  error  in  all  experiments,  with  the  pos- 
sible exception  of  Experiment  2 in  Table  III. 

One  can  attribute  but  a slight  retardation  in  the  rate  of  evapo- 
ration to  ihe  presence  of  the  gelatin,  whereas  the  presence  of 
bacteria,  as  subsequent  experiments  will  reveal,  causes  an  accel- 
eration. The  data  in  Tables  II  and  III  are  rearranged  in  Tables 
IV  and  V to  give  a comparison  between  the  sterile  sets  and  the 
sets  in  which  the  normal  soil  bacteria  were  present  and  per- 
mitted to  develop.  One  finds  here  a uniformity  in  all  cases, 
namely  that  the  normal  sets  all  show  an  increased  rate  of  evapo- 
ration in  contrast  to  the  sterile  sets,  whether  gelatin  was  present 


Relation  of  Soil  Bacteria  to  Evaporation 


189 


TABLE  II.  INFLUENCE  OF  GELATIN  UPON  EVAPORATION 
FROM  STERILE  SOIL 

Sterile  conditions  were  maintained  throughout. 


Interval 

Loss  of  moisture 
in  grams 

Differ- 
ence in 
favor  of 
gelatin 
set 

Total 

Tot  al  loss  of 
moisture  in 
grams 

Total 
differ- 
ence in 

With- 
out gel- 
atin 

With 

gelatin 

period 

With- 
out gel- 
atin 

With 

gelatin 

favor  of 
gelatin 
set 

Experiment  1 

From  start  to  2nd  day.. . . 

5.56 

5.60 

+0.04 

2 days 

5.56 

5.60 

+0.04 

2nd  to  4th  day 

4.54 

4.60 

+0.06 

4 days 

10.10 

10.20 

+0.1 

4th  to  7th  day 

7.80 

7.80 

0.00 

7 days 

17.90 

18.00 

+§.l 

7th  to  9th  day 

14.00 

13.30 

-0.7 

9 days 

31.90 

31.30 

—0.6 

9th  to  13th  day 

20.10 

19.70 

-0.4 

13  days 

52.00 

51.00 

—1.0 

Experiment  2 

From  start  to  14th  hr 

10.67 

10.00 

-0.67 

14  hrs. 

10.67 

10.00 

—0.67 

14th  hr.  to  24th  hr 

19.83 

18.95 

—0.88 

1 day 

30.50 

28.95 

—1.55 

1st  to  3rd  day 

15.00 

16.35 

+1.35 

3 days 

45.50 

45.30 

— 0.2C 

3rd  to  4th  day 

16.00 

15.47 

—0.53  | 

4 days 

61.50 

60.77 

—0.73 

4th  to  5th  day 

14.00 

14.60 

+0.60 

5 days 

75.50 

75.37 

—0.13 

5th  to  7th  day 

18.50 

18.83 

+0.33 

7 days 

94.00 

94.20 

+0.2 

7th  to  9th  day 

33.10 

33.20 

+0.10 

9 days 

127.10 

127.40 

+0.3 

9th  to  12th  day 

39.37 

38.20 

-1.17 

12  days 

166.47 

165.60 

-0.87 

12th  to  14th  day 

16.23 

16.40 

+0.17 

14  days 

182.70 

182.00 

— 0.7C 

TABLE  III.  INFLUENCE  OF  GELATIN  ON  EVAPORATION  FROM  NORMAL 

SOIL 

Soil  under  normal  conditions  with  bacteria  present. 


Loss  of  moisture 
in  grams 

Differ- 
ence in 

Total 

Total  loss  of 
moisture  in 
grams 

Total 
differ- 
ence in 

Interval 

With- 

out 

gelatin 

With 

gelatin 

favor  of 
gelatin 
set 

period 

With- 

out 

gelatin 

With 

gelatin 

favor  of 
gelatin 
set 

Experiment  1 
From  start  to  2nd  day 

6.50 

5.90 

—0.60 

2 days 

6.50 

5.90 

—0.60 

2nd  to  4th  day 

4.60 

5.20 

+0.60 

4 days 

11.10 

11.10 

0.00 

4th  to  7th  day 

8.00 

8.30 

+0.30 

7 days 

19.10 

19.40 

+0.30 

7th  to  9th  day 

13.50 

11.20 

—2.30 

9 days 

32.60 

30.60 

—2.00 

9th  to  13th  day 

21.00 

19.70 

—1.30 

13  days 

53.60 

50.30 

—3.30 

Experiment  2 

From  start  to  14th  hr 

11.00 

11.95 

i +0.95 

14  hrs. . 

11.00 

11.95 

+0.95 

14th  to  24th  hr 

21.56 

20.55 

—1.01 

24  hrs. . 

32.56 

32.50 

-0.06 

1st  to  3rd  day 

17.71 

17.31 

—0.40 

3 days 

50.27 

49.81 

-0.46 

3rd  to  4th  day 

18.73 

18.19 

—0.54 

4 days 

69.00 

68.00 

—1.00 

4th  to  5th  day, 

18.50 

15.50 

—3.00 

5 days 

87.50 

83.50 

—4.00 

5th  to  7th  day 

24.20 

19.90 

—4.30 

7 days 

111.70 

103.40 

—8.30 

7th  to  9th  day 

34.90 

34.00 

— 0.P0 

9 days 

146.60 

137.40 

—9.20 

9th  to  12th  day.  . . 

30.80 

34.10 

+3.30 

12  days 

177.40 

171.50 

-5.90 

12th  to  14th  day 

10.27 

14.30 

+4.03 

14  days 

187.67 

185.80 

—1.87 

190 


Wisconsin  Research  Bulletin  No.  23 


TABLE  IV.  INFLUENCE  OF  BACTERIAL  DEVELOPMENT  UPON  EVAPOR- 
ATION  IN  THE  ABSENCE  OF  GELATIN 
Rearrangement  of  data  given  in  Tables  II  and  III. 


Interval 

Loss  of  moisture 
in  grams 

Differ- 
ence in 
favor  of 
normal 
set 

Sterile 

Normal 

Experiment  1. 
From  start  to  2nd  day. . . 
2nd  to  4th  day 

5.56 

4.54 

7.80 

14.00 

20.10 

6.50 

4.60 

8.00 

13.50 

21.00 

+0.94 
+0.06 
+0.20 
A £A 

4th  to  7th  day 

7th  to  9th  day 

9th  to  13th  day 

— U . DU 

+0.90 

Total 

period 


Total  loss  of 
moisture  in 
grams 


Sterile 


Normal 


Total 
differ- 
ence in 
favor  of 
normal 
set 


2 days 
4 days 
7 days 
9 days 
13  days 


5.56 

10.10 

17.90 

31.90 
52.00 


6.50 

11.10 

19.10 

32.60 

53.60 


+0.94 

+1.00 

+1.20 

+0.70 

+1.60 


Experiment  2. 

From  start  to  14th  hr.... 

14th  to  24th  hr 

1st  to  3rd  day 

3rd  to  4th  day  ... 

4th  to  5th  day 

5th  to  7th  day 

7th  to  9th  day 

9th  to  12th  day.  ... 

12th  to  14th  day 


10.67 

19.83 

15.00 

16.00 
14. 0C 
18.50 
33.10 
39.37 
16.23 


11.00 

21.56 

17.71 

18.73 

18.50 

24.20 

34.90 

30.80 

10.27 


+0.33 

+1.73 

+2.71 

+2.73 

+4.50 

+5.70 

+1.80 

—8.57 

—5.96 


14  hrs. 
24  hrs. 

3 days 

4 days 

5 days 
7 days 
9 days 

12  days 
14  days 


10.67 
30  50 

45.50 

61.50 

75.50 
94.00 

127.10 

166.47 

182.70 


11.00 
32.56 
50.27 
69  00 
87.50 
111.70 
146.60 
177.40 
187.67 


+0.33 
+2.06 
+4.77 
+7.50 
+12.00 
+17.70 
+ 19.50 
+10.93 
+4.97 


TABLE  V.  INFLUENCE  OF  BACTERIAL  DEVELOPMENT  UPON 
EVAPORATION  IN  THE  PRESENCE  OF  GELATIN 

Rearrangement  of  data  given  in  Tables  II  and  III. 


Interval 

Loss  of  moisture 
in  grams 

Differ- 
ence in 
favor  of 

Total 

Total  loss  of 
moisture  in 
grams 

Total 
differ- 
ence in 
favor  of 
normal 
set 

Sterile 

Normal 

normal 

set 

period 

Sterile 

Normal 

Experiment  1 

From  start  to  2nd  day 

2nd  to  4th  day 

4th  to  7th  day 

5.60 

4.60 
7.80 

13.20 

19.70 

5.90 
5.20 
8.30 
1 11.20 
19.70 

+0.30 
+0.60 
A £A 

2 days 
4 days 
7 days 
9 days 
13  days 

5.60 

10.20 

18.00 

31.30 

51.00 

5.90 

11.10 

19.40 

30.60 

50.30 

+0.30 

+0.90 

+1.40 

—0.70 

—0.70 

7th  to  9th  day 

9th  to  13th  day 

i U . DU 
—2.10 
0.00 

E*periment  2 

From  start  to  14th  hr. . 
14th  to  24th  hr 

10.00 

18.95 

16.35 

15.47 

14.60 

18.83 

33.20 

38.20 
16.40 

11.95 

20.55 

17.31 

18.19 

15.50 

19.90 

34.00 

34.10 

11.30 

+ 1.95 
4-i  on 

14  hrs. 

10.00 

28.95 

4S.30 

60.77 

75.37 

94.20 

127.40 

165.60 

182.00 

11.95 
32.50 
49.81 
68.00 
83  50 

103.40 

137.40 
171.50 
185.80 

+1.95 

+3.55 

+4.51 

+7.23 

+8.13 

+9.20 

+10.00 

+5.90 

+3.80 

1st  to  3rd  day 

l 1 . rU 
-LA  QA 

^ ills. 

3 days 

4 days 

5 days 
7 days 
9 days1 
12  days] 
14  days 

3rd  to  4th  day 

4th  to  5th  day 

-+2.72 

4_n  qa  1 

5th  to  7th  day 

i U.UU  ! 

4-1  A7  ' 

7tli  to  9th  day ■ 

l J .Ul 

-La  qa 

9th  to  12th  day 

12th  to  14tli  day 

l U.oU 

—4.10  | 
—2.10  1 

or  not.  It  is  further  interesting  to  note  that  the  differences  in 
the  rate  of  evaporation  between  the  sterile  and  the  normal  sets 
were  reduced,  apparently  by  the  presence  of  gelatin ; a tendency 
as  it  were,  of  neutralizing  the  increased  rate.  This  is  in  har- 
mony with  the  results  shown  in  Tables  II  and  III  where  it  ap- 


Relation  of  Soil  Bacteria  to  Evaporation 


191 


pears  that  gelatin,  probably  clue  to  its  viscid  nature,  exerted  a 
slight  retardation  upon  the  rate  of  evaporation. 

Problem  II.  Influence  of  Soil  Bacteria  on  Rate  of  Evapo- 
ration 

The  influence  of  the  bacterial  flora  of  the  soil  upon  evapora- 
tion was  investigated  before  proceeding  to  any  special  modifi- 
cation of  the  work.  Several  soils  were  thus  examined.  They 
were  air-dried  and  then  passed  through  a twelve-mesh  sieve' and 
thoroughly  mixed.  In  this  way,  a uniform  and  thoroughly  rep- 
resentative sample  was  secured  for  each  plate  prepared.  Eight 
plates  for  each  soil  were  prepared,  using  500-gram  portions. 
To  each  of  four  of  these,  200  c.  c.  distilled  water  were  added  to 
serve  as  a normal  or  inoculated  set.  To  each  of  the  other  four, 
200  c.  c.  of  a 1 :1000  mercuric  chloride  solution3  were  added. 
Thus  the  treatment  of  all  plates  was  identical  except  that  mer- 
curic chloride  was  added  to  the  sterile  sets.  In  the  normal  or 
inoculated  sets,  bacterial  development  occurred ; in  the  sterile 
sets,  it  was  inhibited.  As  has  been  shown  by  preliminary  work, 
the  addition  of  the  mercuric  chloride  solution,  in  the  amounts 
employed,  has  no  appreciable  or  measureable. effect  upon  the  loss 
of  moisture.  Accordingly,  any  differences  which  would  occur 
between  the  inoculated  and  the  sterile  sets  above  mentioned 
must  of  necessity  be  attributed  to  the  bacterial  activities  taking 
place  in  the  inoculated  plates. 

The  results  of  this  series  of  experiments  are  recorded  in  con- 
densed form  in  Tables  VI  to  XI,  the  figures  reported  represent- 
ing the  average  of  the  four  plates  in  each  set.  Data  are  here 
given  showing  the  Joss  of  moisture  for  the  various  daily  inter- 
vals as  well  as  for  the  different  total  periods.  Comparisons  are 
also  made  between  the  sterile  and  the  normal  sets,  the  difference 
between  the  two  for  all  soils  being  indicated.  In  those  cases 
where  the  normal  or  inoculated  sets  showed  a greater  evapora- 
tion than  the  steriles,  the  differences  are  indicated  by  a plus 
sign;  where  the  sterile  exceeded  the  normal  or  inoculated,  a 
minus  sign  is  used  to  indicate  the  difference. 


3 Made  by  adding  20  c.  c.  of  a 1:100  solution  to  180  c.  c.  of  distilled 
water. 


192 


Wisconsin  Research  Bulletin  No.  23 


It  is  at  once  apparent  that  no  constancy  prevailed  in  the  rate 
of  evaporation  from  the  various  soils.  They  revealed  consider- 
able variation,  probably  due  to  differences  in  their  physical  and 
chemical  composition. 


table  vl  influence  of  normal  soil  bacteria  upon 
rate  of  evaporation  from  greenhouse  soil 


• Interval 

Loss  of  moisture 
in  grams 

Differ- 
ence in 
favor  of 
inoc- 
ulated 
set 

Total 

period 

Total 

loss  of  moisture 
in  grams 

Total 
differ- 
ence in 
favor  of 
inoc- 
ulated 
set 

Sterile 

Inoc- 

ulated 

Sterile 

Inoc- 

ulated 

1st  14  hrs 

10.67 

11.00 

+0.33 

14  hrs. 

10.67 

11.00 

+0.33 

14th  to  24th  hr 

19.86 

21.56 

+1.70 

24  hrs. 

30.53 

32.56 

+2.03 

1st  to  3rd  day 

14.97 

17.70 

+2.73 

3 days 

45.50 

50.26 

+4.76 

4th  day 

16.00 

18.70 

+2.70 

4 days 

61.50 

68.96 

+7.46 

5th  day 

14.00 

18.50 

+4.50 

5 days 

75.50 

87.46 

+11.96 

5th  to  7th  day 

18.82 

24.20 

+5.38 

7 days 

94.32 

111.66 

+17.34 

7th  to  9th  day 

33.17 

34.85 

+1.68 

9 days 

127.49 

146.51 

+19.02 

9th  to  12th  day 

39.37 

30.80 

-8.57 

12  days 

166.86 

177.31 

+10.45 

12th  to  14th  day 

16.30 

10.20 

—6.10 

14  days 

183.16 

187.51 

+4.35 

table  VII.  influence  of  normal  soil  bacteria  upon  rate  of 
evaporation  from  white  quartz  sand 


Interval 

Loss  of  moisture 
in  grams 

Differ- 
ence in 
favor  of 
inocu- 
lated 
set 

Total 

per- 

iod 

Total 

loss  of  moisture 
in  grams 

Total 
differ- 
ence in 
favor  of 
inocu- 
lated 
set 

Sterile 

Inocu- 

lated 

Sterile 

' 

Inocu- 

lated 

1st  14  hrs 

9.50 

10.50 

+1.00 

14  hrs. 

9.50 

10.50 

+1.00 

14th  to  24th  hr 

18.75 

22.27 

+3.52 

24  hrs. 

28.25 

32.77 

+4.52 

1st  to  3rd  day 

15.60 

17.50 

+1.90 

3 d ays 

43.85 

50.27 

+5.42 

4th  day 

15.57 

19.45 

+3.88 

4 days 

59.42 

69.72 

+9.30 

5th  day 

15.67 

17.30 

+1 .63 

6 days 

75.09 

87.02 

+10.93 

5th  to  7th  day 

22.00 

25.80 

+3.80 

7 days 

97.09 

112.82 

+ 14.73 

7th  to  9th  day 

19.00 

19.77 

+0.77 

9 days 

116.09 

132.59 

+15.50 

table  viii.  influence  of  normal  soil  bacteria,  upon  rate  of 

EVAPORATION  FROM  CLAY  LOAM  SOIL 


Day  interval 

Loss  of  moisture 
in  grams 

Differ- 
ence in 
favor  of 
inocu- 
lated 
set 

Total 
num- 
ber of 
days 

Total  loss  of 
moisture  in 
grams 

Total 
differ- 
ence in 
favor  of 
inocu- 
lated 
set 

Sterile 

Inocu- 

lated 

Sterile 

Inocu- 

lated 

1st 

28.72 

26.10 

—2.62 

1 

28.72 

26.10  ' 

—2.62 

2nd 

13.45 

14.90 

+1.45 

2 

42.17 

41.00 

—1.17 

3rd 

18.35 

20.(0 

+1.65 

3 

60.52 

61.00 

+0.48 

4th 

15.50 

18.90 

+3.40 

4 

76.02 

79.90 

+3.88 

5th 

15.70 

16.70 

+ 1.00 

5 

91.72 

96.60 

+4.98 

6th 

14.40 

15.77 

+1.37 

6 

106.12 

112.37 

+6.25 

7th 

12.55 

15.15 

+2.60 

7 

118.67 

127.52 

+8.85 

8th 

12.42 

13.40 

+0.98 

8 

131.09 

140.92 

+9.83 

Relation  of  Soil  Bacteria  to  Evaporation 


193 


TABLE  IX.  INFLUENCE  OF  NORMAL  SOIL  BACTERIA  UPON  RATE  OF 
EVAPORATION  FROM  SANDY  SOIL 


Day  interval 

Loss  of  moisture 
in  grams 

Differ- 
ence in 
favor  of 
inocu- 
lated 
set 

| 

1 Total 
num- 
ber 
of 

days 

Total  loss  of 
moisture  in 
grams 

Total 
differ- 
ence in 
favor  of 
inocu- 
lated 
set 

Sterile 

Inocu- 

lated 

Sterile 

Inocu- 

lated 

1st 

29.70 

30.07 

+0.37 

1 

29.70 

30.07 

+£>.37 

2nd 

14.65 

15.72 

+1.07 

2 

44.35 

45.79 

+ 1.44 

3rd 

18.25 

20.00 

+ 1.75 

3 

62.60 

65.79 

+3.19 

4th 

15.47 

17.10 

+1.63 

4 

78.07 

82.89 

+4.82  1 

5th 

18.55 

18.42 

—0.07 

5 

96.62 

101.31 

+4.69 

6th 

17.11 

17.90 

+0.80 

6 

113.72 

119.21 

+5.49 

7th 

15.60 

16.60 

+1.00 

7 

129.32 

135.81 

+6.49 

8th 

14.70 

15.42 

+0.72 

8 

144.02 

151.23 

+7.21 

TABLE  X.  INFLUENCE  OF  NORMAL  SOIL  BACTERIA  UPON  RATE  OF 
EVAPORATION  FROM  FIELD  CLAY  SOIL. 


Day  interval 

Loss  of  moisture 
in  grams 

Differ- 
ence in 
favor  of 
inocu- 
lated 
set 

Total 
num- 
ber of 
days 

Total  loss  of 
moisture  in 
grams 

Total 
differ- 
ence in 
favor  of 
inocu- 
lated 
set 

Sterile 

Inocu- 

lated 

Sterile 

Inocu- 

lated 

1st 

18.0 

15.2 

—2.8 

1 

18.0 

15.2 

—2.8 

2nd 

18.0 

15.7 

-2.3 

2 

36.0 

30.9 

—5.1 

3rd 

21  2 

20.7 

~ — 0 . 5 

3 

57.2 

51.6 

—5.6 

4th 

23  + 

25.5 

+ 2.5 

4 

80.2 

77.1 

—3.1 

5th 

23.0 

25.0 

+2.0 

5 

105.2 

102.1 

—1.1 

6th 

20.1 

24.2 

I +4.1 

6 

123.3 

126.3 

+3.0 

7th 

19.6 

23.4 

+3.8 

7 

142.9 

149.7 

+6.8 

8th 

12.2 

16.5 

+4.3 

8 

155.1 

166.2 

+11.1 

9th 

14.6 

14.8 

+0.2 

9 

169.7 

181.0 

+ 11.3 

10th 

13.3 

12.7 

—0.6 

10 

183.0 

193.7 

+10.7 

11th 

5.8 

4.2 

1 .6 

11 

188.8 

197.9 

+9.1 

12th 

1.5 

1.3 

—0.2 

12 

190  3 

199.2 

+8.9 

13th 

1.2 

1.2 

0.0 

13 

191.5 

200.4 

+8.9 

TABLE  XI.  INFLUENCE  OF  NORMAL  SOIL  BACTERIA  UPON  RATE  OF 
EVAPORATION  FROM  MUCK  SOIL 


Day  interval 

Loss  of  moisture 
in  grams 

Differ- 
ence in 
favor  of 
inocu- 
lated 
set 

Total 
num- 
ber of 
days 

Total 

loss  of  moisture 
in  grams 

Total 
differ- 
ence in 
favor  of 
inocu- 
lated 
set 

Sterile 

Inocu- 

lated 

Sterile 

Inocu- 

lated 

1st  to  2nd 

33  40 

33.80 

+0.40 

2 

33.40 

33.80 

+0.40 

2nd  to  4th 

40.10 

44.50 

+4.40 

4 

73.50 

78.30 

+4.80 

5th  day 

19.40 

20.00 

. +0.60 

5 

92.90 

98.30 

+5.40 

5th  to  7th 

26.20 

26.10 

—0.10 

7 

119.10 

124.40 

+5.30 

7th  to  9th 

21.49 

21.30 

— 0-19 

9 

140.59 

145.70 

+5.11 

9th  to  12th 

21 .67 

20.75 

— 0.82 

12 

162.16 

166.45 

+4.29 

12th  to  14th 

9.60 

8.45 

— 1.15 

14 

171.76 

174.90 

+3.14 

194 


Wisconsin  Research  Bulletin  No.  23 


One  finds  the  maximum  interval  variation  between  sterile  and 
normal  sets  in  the  greenhouse  and  the  field  clay  soils,  and  the 
least  variation  with  the  sandy  soil.  On  total  variations  the 
greenhouse  soil  exceeds  all  others  showing  as  a maximum,  19.02 
grams  difference  in  favor  of  the  inoculated  set.  It  is  interesting 
to  note  that  in  all  cases,  the  rate  of  evaporation  was  more  rapid 
in  the  inoculated  set  than  in  the  sterile  set. 

This  difference  in  favor  of  the  inoculated  plates  is  maintained 
with  all  the  soils  examined,  and  furthermore  is  so  large  as  to  ex- 
ceed the  factor  of  error. 

A condition  which  is  here  evident,  and  which  will  become 
more  so  as  the  discussion  of  subsequent  experiments  progresses, 
is  the  fact  that  as  the  plates  approach  an  air-dry  condition  the 
steriles  show  increased  evaporation  in  contrast  to  the  normal  or 
inoculated  plates;  a more  rapid  rate  of  evaporation  seems  to 
occur  in  the  steriles,  and  ultimately  the  total  evaporation  for 
both  sets  is  again  equal.  This  is  due  to  the  fact  that  the  nearer 
air-dry  the  soil  becomes,  the  more  tenaciously  the  water  is  held. 
This  condition  is  reached,  as  is  apparent  from  the  data  in  these 
and  all  other  experiments,  about  the  ninth  day,  from  which  time 
the  total  differences  in  favor  of  the  inoculated  set,  begin  to  show 
a gradual  decline.  With  the  more  rapid  evaporation  in  the  in- 
oculated set  during  the  earlier  part  of  the  experiment  the  air- 
dry  condition  is  more  rapidly  approached,  after  which  evapora- 
tion is  much  slower  than  in  the  sterile  set  which,  due  to  the 
slower  evaporation  in  the  earlier  stages,  does  not  approach  the 
air-dry  condition  so  rapidly.  Another  feature  worthy  of  men- 
tion is  the  fact  that  the  greatest  variations  for  the  daily  periods, 
between  the  sterile  and  inoculated  sets  usually  occur  between 
the  fourth  and  sixth  days  of  the  experiments.  It  is  well  to  bear 
this  fact  in  mind,  as  subsequent  experiments  show  that  it  is  dur- 
ing this  period  that  bacterial  activity  is  greatest.  The  maximum 
interval  variations  in  favor  of  the  inoculated  series  coincide 
more  or  less  closely  with  the  period  of  greatest  bacterial  activity. 
As  this  condition  holds  for  all  soils  experimented  upon,  one  can 
justly  conclude  that  the  increased  rate  of  evaporation  in  the 
inoculated  series  must  be  attributed  to  the  bacteria  themselves 
or  to  changes  in  the  physical  and  chemical  composition  of  the 
soil,  occasioned  by  their  activity.  That  this  increase  may  be 
considerable  has  already  been  pointed  out,  amounting  in  the 


Relation  of  Soil  Bacteria  to  Evaporation  195 

case  of  the  greenhouse  soil  to  19.02  grams,  which  is  approxi- 
mately 10%  of  the  total  moisture  originally  added. 

The  question  naturally  arises,  “ Do  these  conditions  occur  in 
the  field  ? ’ ’ Such  wide  differences  would  hardly  be  possible,  but 
it  must  be  admitted  that  bacteria  can  and  undoubtedly  do  in- 
fluence the  rate  of  evaporation  by  affecting  the  movement  of 
soil  water.  To  what  extent,  cannot  be  said  at  this  time.  What- 
ever influence  is  exerted  has  probably  been  magnified  by  the 
nature  of  the  experiments  thus  far  conducted. 

Problem  III.  Relation  Between  Bacterial  Multiplication 
and  Increased  Evaporation 

The  fact  that  the  maximum  interval  variation  between  the 
sterile  and  inoculated  series  usually  occurred  between  the  fourth 
and  sixth  days,  rendered  it  advisable  to  determine  experimen- 
tally whether  any  relation  existed  between  the  maximum  loss 
of  moisture  and  the  highest  bacterial  development.  With  this 
end  in  view  the  following  experiments  were  undertaken: 

Two  sets  of  six  plates  each  were  prepared,  identical  in  every 
respect  except  that  to  one  set  200  c.  c.  of  a 1 :1000  solution  of 
mercuric  chloride  per  plate  had  been  added.  Each  plate  of  the 
other  set  received  200  c.  c.  of  distilled  water.  All  of  the  plates 
were  then  exposed.  Upon  three  of  the  plates  of  each  set,  mois- 
ture and  bacterial  determinations  were  made  daily,  the  germ 
content  being  then  reduced  to  the  dry  basis.  The  other  three 
plates  of  each  set  were  left  undisturbed,  but  were  weighed  daily 
to  ascertain  the  rate  of  the  loss  of  moisture.  In  this  way  data 
were  secured  giving  the  daily  loss  of  moisture  and  the  germ 
content  of  the  soils  on  the  respective  days.  It  was  thus  possible 
to  make  a direct  comparison  between  the  loss  of  moisture  due  to 
evaporation,  and  the  germ  content  of  the  soil.  These  experi- 
ments were  repeated  employing  a dilute  bouillon  solution  for 
moistening  purposes  in  place  of  the  ordinary  distilled  water 
which  had  been  employed  in  all  previous  experiments.  It  was 
thought  that  the  addition  of  this  bouillon  would  enhance  bac- 
terial activity  to  a large  extent  and  perhaps  magnify  the  differ- 
ences in  the  rate  of  evaporation.  All  data  in  connection  with 
these  two  experiments  are  reported  in  Tables  XII  and  XIII. 
These  reveal  the  fact  observed  in  the  previous  experiments,  that 


196 


Wisconsin  Research  Bulletin  No.  23 


TABLE  XII.  RELATION  BETWEEN  BACTERIAL  COUNT  AND  EVAPORATION 
Distilled  water  used  to  moisten  soil.  Ordinary  soil  infusion  used  for  inoculation. 


Day 

interval 

Bacter- 
ial count 
in  mil- 
lions pei 
gram 

Loss  of  moisture 
in  grams 

Differ- 
ence in 
favor  of 
inoc- 
ulated 
set 

Total 
num- 
ber of 
days 

Loss  of  moisture 
in  grams 

Differ- 
ence in 
favor  of 
inoc- 
ulated 
set 

Sterile 

Inoc- 

ulated 

Sterile 

Inoc- 

ulated 

Soil  infus- 

ion 

No.  1. 

1st 

2.6 

19.76 

19.70 

—0.06 

1 

19.76 

19.70 

—0.06 

2nd 

20.60 

23.90 

+3.30 

2 

40.36 

43.60 

+3.36 

3rd 

12.45 

22.30 

23.80 

+1.50 

3 

62.66 

67.40 

+4.86 

4th 

20.90 

21.30 

25.60 

+4.30 

4 

83.96 

93.00 

+9.16 

5th 

45.80 

13.30 

18.00 

+4.70 

5 

97.26 

111.00 

+13.86 

6th 

35.45 

13.90 

20.20 

+6.30 

6 

111.16 

131.20 

+20.16 

7th 

35.40 

14.00 

18.50 

+4.50 

7 

125.16 

149.70 

+24.66 

8th 

30.20 

12.50 

15.00 

+2.50 

8 

137.66 

164.70 

+27.16 

Soil  infus- 

ion 

No.  2. 

1st 

5.3 

18.0 

15.2 

—2.8 

1 

18.0 

15.2 

—2.8 

2nd 

9.7 

18.0 

15.7 

—2.3 

2 

36.0 

30.9 

—5.1 

3rd 

18.7 

21.2 

20.7 

-0.5 

3 

57.2 

51.6 

—5.6 

4th 

10.8 

23.0 

25.5 

+2.5 

4 

80.2 

77.1 

—3.1 

5th 

9.0 

23.0 

25.0 

+2.0 

5 

103.2 

102.1 

— l.l 

6th 

8.3 

20.1 

24.2 

+4.1 

6 

123.3 

126.3 

+3.0 

7th 

6.3 

19.6 

23.4 

+3.8 

7 

142.9 

149.7 

+6.8 

8th 

5.6 

12.2 

16.5 

+4.3 

8 

155.1 

106.2 

+11.1 

9th 

4.5 

14.6 

14.8 

+0.2 

9 

169.7 

181.0 

+11.3 

10th 

4.6 

13.3 

12.7 

-0.6 

10 

183.0 

193.7 

+10.7 

11th  . . . ? . . . 

5.8 

4.2 

—1.6 

11 

188.8 

197.9 

+9.1 

12th 

1.5 

1.3 

—0.2 

12 

190.3 

199.2 

+8.9 

13th 

1.2 

1.2 

0.0 

13 

191.5 

200.4 

+8.9 

TABLE  XIII.  RELATION  BETWEEN  BACTERIAL  COUNT  AND 
EVAPORATION 


Dilute  bouillon  used  to  moisten  soil.  Ordinary  soil  infusion  used  for  inoculation. 


Day 

interval 

Bacter- 
ial count 

Loss  of  moisture 
in  grams 

Differ- 
ence in 
favor  of 
inocu- 
lated 
set 

Total 

in  mill- 
ions per 
gram 

Sterile 

Inocu- 

lated 

num- 
ber of 
days 

1st 

3.5 

19.6 

18.2 

—1.4 

1 

2nd 

35.0 

17.6 

16.7 

—0.9 

2 

3rd 

1500.0 

17.4 

19.1 

+1.7 

3 

4 th 

10000.0 

17.0 

15.2 

-1.8 

4 

5th 

10000.0 

19.8 

20.0 

+0.2 

5 

6th 

8000.0 

16.8 

18.0 

+1.2 

6 

7th  

8000.0 

17.6 

20.2 

+2.6 

7 

8th 

7560.0 

13.9 

14.9 

+1.0 

8 

9th 

6000.0 

15.5 

18.1 

+2.6 

9 

10th 

3400.0 

14.0 

16.0 

+2.0 

10 

11th 

175.0 

14.2 

19.5 

+5.3 

11 

12th 

20.0 

14.2 

17.7 

+3.5 

12 

13th 

12.8 

10.8 

10.1 

—0.7 

13 

14th 

7.0 

7.9 

7.6 

—0.3 

14 

15th 

9.9 

7.4 

—2.5 

15 

Loss  of  moisture 
in  grams 


Sterile 


19.6 
37.2 

54.6 

71.6 
91.4 

108.2 

125.8 

139.7 

155.2 

169.2 

183.4 
197.6 

208.4 

216.3 
226.2 


Inocu- 

lated 


18.2 

34.9 

54.0 

69.2 

89.2 

107.2 
127.4 

142.3 

164.4 

176.4 
195.9 

213.6 

223.7 
231.3 

238.7 


Differ- 
ence in 
favor  of 
inocu- 
lated 
set 


—1.4 

—2.3 

—0.6 

—2.4 

—2.2 

—0.2 

+2.4 

+3.4 

+6.0 

+8.0 

+13.3 

+16.8 

+16.1 

+15.8 

+13.3 


Delation  of  Soil  Bacteria  to  Evaporation  i9Y 

the  maximum  daily  differences  invariably  occurred  between  the 
fourth  and  sixth  days  and  that  these  maximum  differences  run 
more  or  less  parallel  to  the  maximum  germ  contents  of  the  soils, 
or  immediately  follow  them.  As  was  to  be  expected  in  the  case 
where  dilute  bouillon  had  been  employed,  a most  marked  bac- 
terial multiplication  occurred,  amounting  on  the  fourth  and  fifth 
days  to  over  10,000,000,000  per  gram  of  soil,  but  in  this  case 
such  parallelism  between  maximum  daily  variation  and  maxi- 
mum germ  content  was  not  found.  The  extensive  bacterial  de- 
velopment probably  fixed,  so  to  speak,  a large  percentage  of  the 
moisture  in  the  protoplasm  of  the  bacterial  cells,  holding  and 
preventing  it  from  evaporating.  In  the  other  cases,  where  the 
bacterial  multiplication  was  not  so  extreme,  we  find  a close 
parallelism  between  maximum  germ  content  and  maximum  in- 
terval variation,  a fact  which  apparently  indicates  that  the  bac- 
terial activities  are  responsible  for  the  increased  rate  of  evapo- 
ration in  the  normal  or  inoculated  series. 

Problem  IV.  Effect  of  Kemoistening  on  Increased  Evapo- 
ration 

In  an  endeavor  to  demonstrate  still  further  the  part  played 
by  the  bacteria  in  causing  the  increased  rate  of  evaporation,  a 
slight  modification  of  the  previous  experiments  was  performed 
by  restoring  to  their  original  weights  several  series  of  plates 
after  they  had  been  exposed  to  evaporation.  It  was  thought 
that  thus  the  initial  number  of  bacteria  present  would  be  greater, 
and  that  accordingly  a more  active  evaporation  would  occur. 

On  comparing  the  data  given  in  Table  XIV  one  finds  in  every 
case  a decided  increase  in  the  total  rate  of  evaporation,  over  the 
increase  shown  in  the  initial  experiment,  even  where  modified 
by  the  different  treatments  as  indicated. 

A greater  rate  of  evaporation  in  the  inoculated  series  after 
remoistening  than  initially,  is  to  be  noted  in  all  three  experi- 
ments. These  substantiate  the  evidence  that  bacteria  are  re- 
sponsible directly  or  indirectly  for  the  increased  evaporation 
noted  in  all  cases  in  the  normal  soils.  While  no  germ  content 
determinations  were  made  upon  the  soils  before  and  after  re- 
moistening,  it  is  reasonable  to  suppose  that  the  germ  content  was 
greater  after  remoistening  than  prior  thereto.  With  this  prob- 
able greater  bacterial  content,  a greater  difference  in  rate  of 
evaporation  was  observed.  It  is  further  interesting  to  note  that 


198 


Wisconsin  Research  Bulletin  No.  23 


the  maximum  daily  difference  in  favor  of  the  inoculated  set  falls 
between  the  fifth  and  sixth  days,  results  which  are  in  harmony 
with  the  previous  experiments. 


TABLE  XIV.  INCREASED  EVAPORATION  DUE  TO  REMOISTENING 


Day 


1st 

2nd  to  4th . . 
4th  to  5th.. 
5th  to  7th.. 
7th  to  9th.. 
9th  to  12th. 
12th  to  14th 


interval 


Interval  increase  of 
normal  over  sterile 
expressed  in  grams 


Initially 


After  re- 
moisten- 
ing 


Total 
number 
of  days 


Total  increase  of 
normal  over  sterile 
expressed  in  grams 


Initially 


After  re- 
moisten- 
ing 


+2.1 

+3.8 

+2.2 

+0.1 

—0.4 

—3.2 

—1.2 


—0.9 

+4.3 

+2.9 

+3.7 

+4.7 


1 

4 

5 
7 
9 

12 

14 


+2.1 

+5.9 

+8.1 

+8.2 

+7.8 

+4.6 

+3.4 


—0.9 

+3.4 

+6.3 

+10.0 

+14.7 


1st 

2nd  to  4th.. 
4th  to  5th.. 
5th  to  7th.. 
7th  to  9th. . 
9th  to  12th. 
12th  to  14th 


+1.0 

+7.0 

+4.0 

0.0 

+0.6 

—7.1 

—1.5 


+2.6 

+4.4 

+3.0 

+6.4 

+0.1 


1 

4 

5 
7 
9 

12 

14 


+1.0 
+8.0 
+ 12.0 
+ 12.0 
+12.6 
+5.5 
+4.0 


+2.6 
+7.0 
+ 10.0 
+ 16.4 
+16.5 


i 

1st 

—1.8 

+5.3 

+3.3 

—2.3 

0.0 

—4.0 

+0.5 

+0.8 

+1.3 

+4.2 

+4.3 

+1.5 

4 

5 
7 
9 

12 

14 

—1.8 

+3.5 

+6.8 

+4.5 

+4.5 

+0.5 

+1.0 

+0.8 

+2.1 

+6.3 

+10.6 

+12.1 

2nd  to  4th 

4th  to  5th 

5th  to  7th 

7th  to  9th 

9th  to  12th 

12th  to  14th 

Problem  V.  Influence  of  Organic  Matter  on  Evaporation 

It  will  be  remembered  that  the  work  on  the  normal  soil  bac- 
teria was  performed  with  different  types  of  soil  as  substrata 
from  which  evaporation  was  to  take  place,  and  that  upon  these 
the  amount  of  increased  evaporation  due  to  the  presence  of  bac- 
teria varied  with  the  type  of  soil  employed.  For  example,  the 
increased  rate  of  evaporation  from  a sandy  soil  was  not  as  great 
as  that  from  a clay  or  greenhouse  soil.  These  variations  are 
probably  due  to  differences  in  composition  of  the  soils,  both 
physical  and  chemical.  That  the  presence  or  absence  of  organic 
matter  is  important  irom  the  physical  standpoint  in  determin- 
ing texture,  water-holding  capacity,  and  capillarity  of  soils  is 
known.  It  would  seem  that  its  presence  would  also  influence 
the  results  of  these  experiments. 

What  effect  the  addition  of  varying  amounts  of  organic  mat- 


Relation  op  Soil  Bacteria  to  Evaporation 


i99 


ter  to  a given  soil  would  have,  was  not  known,  and  was  thought 
worthy  of  investigation.  For  this  purpose  the  following  ex- 
periments were  conducted. 

Two  groups  of  three  series  each  were  prepared;  one  group 
was  kept  sterile  in  the  usual  manner,  the  other  was  inoculated 
with  a soil  infusion.  The  series  in  each  group  received  2%, 
1%,  and  no  blood  meal  respectively.  After  proper  moistening 
all  were  exposed  under  uniform  conditions  and  the  losses  oc- 
curring  due  to  evaporation  recorded. 

TABLE  XV.  INFLUENCE  OP  BLOOD  MEAL  ON  EVAPORATION  FROM  SOIL 

Changes  in  rate  of  evaporation  caused  by  the  addition  of  varying  amounts  of  blood 
meal  to  clay  loam  soil. 


Day  interval 

Loss  of  moisture 
in  grams 

Differ- 
ence in 
favor  of 
inocu- 
lated 
set 

Total 
num- 
ber of 
days 

Loss  of  moisture 
in  grams 

Differ- 
ence in 
favor  of 
inocu- 
lated 
set 

Sterile 

Inocu- 

lated 

Sterile 

Inocu- 

lated 

Series  I 

No  blood  meal 

1st  to  2nd 

29.1 

31.2 

+2.1 

2 

29.1 

31.2 

+2.1 

2nd  to  4th 

35.4 

39.2 

+3.8  ! 

4 

64.5 

70.4 

+5.9 

5th 

21.2 

23.4 

+2.2 

5 

85.7 

93.8 

+8.1 

5th  to  7th 

31.7 

31.8 

+0.1 

7 

117.4 

125.6 

+8.2 

7th  to  9th 

35.6 

35.2 

-0.4 

9 

153.0 

160.8 

+7.8 

9th  to  12th 

28.3 

25  1 

-3.2 

12 

181.3 

185.9 

+4  6 

12th  to  14th 

8.3 

7.1 

-1.2  1 

14 

189.6 

193.0 

+3.4 

Series  II 

1%  blood  meal 

1st  to  2nd 

28.1 

29.1 

+1.0 

2 

28.1 

29.1 

+1.0 

2nd  to  4th 

36.0 

43.0 

+7.0 

4 

64.1 

72.1 

+8.0 

5 th 

20.3 

24.3 

+4.0  1 

5 

84.4 

96.4 

+12.0 

5th  to  7th 

34.3 

34.3 

0.0  , 

7 

118.7 

130.7 

+12.0 

7 th  to  9th 

31.0 

31.6 

+0.6 

9 

149.7 

162.3 

+12.6 

9th  to  12th 

28.4 

21.3 

— 7.1 

12 

178.1 

183.6 

+5.5 

12  th  to  14  th 

8.7 

7.2 

— 1.5 

14 

186.8 

190.8 

+4.0 

Series  III 

2%  blood  meal 

1st  to  2nd 

31.5 

29.7 

-1.8 

2 

31.5 

29.7 

—1  8 

2nd  to  4th 

37.3 

42.6 

+5.3 

4 

68.8 

72.3 

+3.5 

5th 

21.6 

24.9 

+3.3 

5 

90.4 

97.2 

+ 6.8 

5th  to  7th 

33.8 

31.5 

-2.3 

7 

124.2 

128.7 

+4.5 

7th  to  9th 

31.7 

31.7 

0.0 

9 

155.9 

160.4 

+4.5 

9th  to  12th 

25.5 

21.5 

—4.0 

12 

181.4 

181.9 

+0.6 

12th  to  14th 

8.1 

8.6 

+0.5 

14 

189.5 

190.5 

+1.0 

From  the  data  submitted  in  Tables  XV  and  XVI  it  is  seen 
that  the  maximum  difference  between  the  inoculated  and  sterile 
sets  for  any  given  period  was  obtained  in  the  series  containing 
1 °/°  blood  meal.  This  held  true  after  remoistening  the  plate§ 
and  again  subjecting  them  to  evaporation.  In  the  presence  of 
2%  blood  meal  the  difference  in  favor  of  the  inoculated  series 
is  not  so  marked.  The  data  showing  the  total  increased  evap- 
oration due  to  inoculation  for  the  three  series  are  given  in 
Table  XVII. 


200  Wisconsin  Research  Bulletin  No.  23 


At  the  close  of  the  experiments  with  blood  meal  all  plates 
were  restored  to  their  original  weight  by  the  addition  of  water 
and  then  subjected  to  evaporation  a second  time.  The  results 
are  indicated  in  Table  XVI. 

TABLE  XVI.  INFLUENCE  OF  BLOOD  MEAL  ON  EVAPORATION  AFTER 

REMOISTENING 


Day  interval 


Loss  of  moisture 
in  grams 


Sterile 


Inocu- 

lated 


Differ- 
ence in 
favor  of 
inocu- 
lated 
set 


Total 

num- 

ber 

of 

days 


Loss  of  moisture 
in  grams 


Sterile 


Inocu- 

lated 


Differ- 
ence in 
favor  of 
inocu- 
lated 
set 


Series  I 


No  blood  meal 


1st 

13.7 

12.8 

—0.9 

1 

13.7 

12.8 

— 0.9 

2nd 

17.6 

21.9 

+4.3 

2 

31.3 

34.7 

+ 3.4 

3rd 

12.9 

15.8 

+2.9 

3 

44.2 

50.5 

+ 6.3 

3rd  to  5th., 

34.4 

38.1 

+3.7 

5 

78.6 

88.6 

+10.0 

5th  to  8th 

38.6 

43.3 

+4.7 

8 

117.2 

131.9 

+14.7 

Series  II 
1%  blood  meal 
1st 

13.7 

16.3 

+2.6 

1 1 

13.7 

16.3 

+ 2.6 

2nd 

17.8 

22.2 

+4.4 

2 

31.5 

38.5 

+ 7.0 

3rd 

13.7 

16.7 

+3.0 

3 

45.2 

55.2 

+10.0 

3rd  to  5th 

32.7 

39.1 

+6.4 

5 

77.9 

94.3 

+ 16.4 

5th  to  8th 

40.6 

40.7 

+0,1 

1 8 

118.5 

135.0 

+16.5 

Series  III 
2%  blood  meal 


1st 

13.1 

13.9 

+0.8 

1 

13.1 

13.9 

+ 0.8 

2nd 

19.4 

1 20.7 

+1.3 

2 

32.5 

34.6 

+ 2.1 

3rd 

13.5 

17.7 

+4.2 

3 

46.0 

52.3 

+ 6.3 

3rd  to  5th 

34.8 

39.1 

+4.3 

5 

80.8 

91.4 

+10.6 

5th  to  8th 

40.0 

41.5 

+1.5 

8 

120.8 

132.9 

+12.1 

The  comparison  of  results  before  and  after  remoistening 
given  in  Table  XVII,  reveals  close  similarity.  In  both,  Series 
II  shows  the  maximum  difference  in  rate  of  evaporation,  where- 
as Series  III  containing  2%  blood  meal  does  not  show  a differ- 


TABLE  XVII.  TOTAL  INCREASE  IN  GRAMS  IN  EVAPORATION  DUE  TO 
INOCULATION  IN  THE  THREE  BLOOD  MEAL  SERIES 


Number  of  days 

Series  I 
No  blood  meal 

Series  II 
1%  blood  meal 

Series  III 
2$  blood  meal 

2 

Before  remoistening 

+ 2.1 

+ 1.0 

— 1.8 

4 

+ 5.9 

+ 8.0 

+ 3.5 

5 

+ 8.1 

+12.0 

+ 6.8 

7 

+ 8.2 

+12.0 

+ 4.5 

9 

+ 7.8 

+12.6 

+ 4.5 

12 

+ 4.6 

+ 5.5 

+ 0.5 

14  

+ 3.4 

+ 4.5 

+ 1.0 

After  remoistening 

— 0.9 

+ 2.6 

+ 0.8 

2 

- + 3.4 

+ 7.0 

+ 2.1 

3 

+ 6.3 

+10.0 

+ 6.3 

5 

+10.0 

+16.4 

+10.6 

8 

+14.7 

+16.5 

+12.1 

• 

Delation  of  Soil  Bacteria  to  Evaporation  201 

ence  so  great  as  Series  I which  received  no  blood  meal.  Atten- 
tion has  already  been  called  to  the  fact  that  the  differences 
are  greater  after  remoistening  than  before. 

A repetition  of  the  experiment  with  blood  meal,  but  substi- 
tuting barnyard  manure  (dried  and  ground  up  fine  so  as  to  pass 
through  a twelve-mesh  sieve)  gave  the  results  indicated  in 
Table  XVIII  and  summarized  in  Table  XIX. 


table  xviii.  effect  of  barnyard  manure  on  evaporation 

Varying  amounts  of  finely  ground  manure  were  added  to  clay  loam  soil,  soil 
infusion  serving  as  inoculating  material. 


Day 

Loss  of  moisture 
in  grams 

Differ- 
ence in 
favor  of 

Total 

Loss  of  moisture 
in  grams 

Differ- 
ence in 
favor  of 
inocu- 
lated 
set 

interval 

Sterile 

Inocu- 

lated 

inocu- 

lated 

set 

num- 
ber of 
days 

Sterile 

Inocu- 

lated 

Series  I. 
No  manure 

1st  to  2nd 

32.80 

36.90 

+4.10 

2 

32.80 

36.90 

+4.10 

2nd  to  4th 

46.43 

47.46 

+1.03 

4 

79.23 

84.36 

+5.13 

5th 

21.96 

25.80 

+3.84 

5 

101.19 

110,16 

+8.97 

5th  to  7th 

30.93 

33.66 

+2.73 

7 

132.12 

143.82 

+11.70 

7th  to  9th 

34.26 

33.26 

—1.00 

9 

166.38 

177.08 

+10.70 

9th  to  12th 

22.43 

15.30 

—7.13 

12 

188.81 

192  38 

+3.57 

12th  to  14th 

2.56 

1.43 

—1.13 

14 

191.37 

193.81 

+2.44 

Series  II. 
1%  manure 

1st  to  2nd 

36.20 

34.66 

—1.54 

2 

36.20 

34.66 

—1.54 

2nd  to  4th 

43.76 

40.73 

— 3.03 

4 

79.96 

75.39 

—4.57 

5th 

25.66 

23.76 

— 1 .90 

5 

105.62 

99.15 

—6.47 

5th  to  7th 

33.20 

34.06 

+0.86 

7 

138.82 

133.21 

—5.61 

7th  to  9th 

32.06 

31.86 

+0.20 

9 

170.88 

165.07 

—5.81 

9th  to  12th 

21.20 

22.90 

i +1.70 

12 

192.08 

187.97 

—4.11 

12th  to  14th 

2.56 

3.90 

+1.34 

14 

194.64 

191.81 

-2.83 

Series  III. 
2%  manure 

1st  to  2nd 

35.43 

35.66 

+0.23 

2 

35.43 

35.66 

+0.23 

2nd  to  4th 

46.10 

42.86 

-3.24 

4 

81.53 

78.52 

—3.01 

5th 

22.80 

25.40 

+2.60 

5 

104.33 

103.92 

—0.41 

5th  to  7th 

35.03 

33.00 

—2.03 

7 

139.36 

136.92 

—2.44 

7th  to  9th 

32.16 

32.90 

+0.74 

9 

171.52 

169.82 

—1.70 

9th  to  12th 

19.00 

19.96 

+0.96 

12 

190.52 

189.78 

—0.74 

12th  t©  14th 

2.96 

3.10 

+0.14 

14 

193.48 

192.88 

—0.60 

TABLE  XIX.  INFLUENCE  OF  MANURE  ON  TOTAL  EVAPORATION 
Difference  between  inoculated  and  sterile  sets  expressed  in  grams. 


Series  I 

Series,  II 

Series  HI 

Number  of  days 

No  manure 

1%  manure 

2%  manure 

2. 

+4.10 

—1.54 

+0.23 

+5.13 

—4.57 

—3.01 

+8.97 

-6.47 

—0.41 

7. 

+11.70 

—5.61 

-8.44 

9-r 

+10.70 

—5.81 

—1.70 

12.  i 

+3.57 

-4.11 

—0.74 

i4.: 

+2.44 

—2.83 

—0.60 

202 


Wisconsin  Research  Bulletin  No.  23 


TABLE  XX.  INFLUENCE  OF  ADDITION  OF  BLOOD  MEAL 


Day  interval 


Series  I 
No  blood  meal 

1st 

2nd 

3rd  & 4th 

5th 

6th 

7th 

8th 

9th 

10th  & 11th 

12th  & 13th 

14th  & 15th 

16th,  17th  & 18th 

Series  II 
-FI#  blood  meal 

1st 

2nd 

3rd  & 4th 

5th 

6th 

7th 

8th 

9th 

10th  & 11th 

12th  & 13th 

14th  & 15th 

16th,  17th  & 18th 

Series  III 
+ 2%  blood  meal 

1st 

2nd 

3rd  & 4th 

5th 

6th 

7 th 

8th 

9th 

10th  & Uth 

12th  & 13th 

14th  & 15th 

16th,  17th  & 18th 

Series  IV 
+ 3.5%  blood  meal 

1st 

2nd 

3rd  & 4th 

5th 

6th 

7th 

8th 

9th 

10th  & 11th 

12th  & 13th 

14th  & 15th 

16th,  17th  & 18th 

Series  V 
+ 5%  blood  meal 

1st 

2nd 

3rd  & 4th 


5th 

6th 

7th 

8th 

9th 

10th  & 11th 

12th  & 13th 

14th  & 15th 

16th,  17th  & 18th. 


Loss  of  moisture 
in  grams 


Sterile 


Inocu- 

lated 


Differ- 

ence in 

Total 

favor  of 

num- 

inocu- 

ber of 

lated 

days 

set 

Loss  of  moisture 

Differ- 

in grams 

ence  in 
favor  of 
inocu- 
lated 
set 

Sterile 

Inocu- 

lated 

10.6 

10.5 

-0.1 

1 

10.6 

10.5 

—0.1 

6.2 

5.8 

—0.4 

2 

16.8 

16.3 

—0.5 

16.6 

17.3 

+0.7 

4 

33.4 

33.6 

+0.2 

8.8 

8.6 

-0.2 

5 

42.2 

42.2 

0.0 

8.5 

9.6 

+1.1 

6 

50.7 

51.8 

+1.1 

9.6 

11.2 

+1.6 

7 

60.3 

63.0 

+2.7 

6.6 

6.8 

+0.2 

8 

66.9 

69.8 

+2.9 

8.3 

8.6 

+0.3 

9 

75.2 

78.4 

+3.2 

16.8 

17.8 

+1.0 

11 

92.0 

96.2 

+4.2 

13.5 

14.0 

+0.5 

13 

105.5 

110.2 

+4.7 

13.0 

15.2 

+2.2 

15 

118.5 

125.4 

+6.9 

21.0 

24.5 

+3.5 

18 

139.5 

149.9 

+10.4 

10.6 

10.6 

0.0 

! 

10.6 

10.6 

0.0 

6.5 

6.2 

—0.3 

2 

17.1 

16.8 

—0.3 

16.2 

17.0 

+0.8 

4 

33.3 

33. 8 

+0.5 

8.6 

9.0 

+0.4 

5 

41.9 

42.8 

+0.9 

8.8 

9.0 

+0.2 

6 

50.7 

51.8 

+1.1 

9.6 

10.6 

+ 1.0 

7 

60.3 

62.4 

+2.1 

6.6 

6.8 

+0.2 

8 

66  9 

69.2 

+2.3 

8.0 

8.6 

+0.6 

9 

74.9 

77.8 

+2.9 

16.2 

19.0 

+2.8 

11 

91.1 

96.8 

+5.7 

13.0 

14.3 

+1.3 

13 

104.1 

111.1 

+7.0 

13.0 

14.0 

+1.0 

15 

117.1 

125.1 

+8.0 

22.6 

24.2 

+1.6 

18 

139.7 

149.3 

+9.6 

12.2 

11.5 

—0.7  1 

1 

12.2 

11.5 

—0.7 

6.2 

6.5 

+0.3 

2 

18.4 

18.0 

—0.4 

15.5 

16.3 

+0.8 

4 

33.9 

34.3 

+0.4 

8.5 

9.0 

+0.5  1 

5 

42.4 

43.3 

+0.9 

10.0 

9.6 

-0.4  1 

6 

52.4 

52.9 

+0.5 

9.8 

9.5 

-0.3 

7 

62.2 

62.4 

+0.2 

7.3 

7.6 

+0.3 

8 

69.5 

70.0 

+0.5 

8.0 

8.8 

+0.8 

9 

77.5 

78.8 

+1.3 

17.8 

21.2 

+3.4 

11 

95.3 

100.0 

+4.7 

14.0 

16.2 

+2  2 

13 

109.3 

116.2 

+6.9 

13.2 

15.8 

+2.6 

15 

122.5 

132.0 

+9.5 

23.0 

27.0 

+4.0 

18 

145.5 

159.0 

+13.5 

11.5 

11.8 

+0.3 

1 

11.5 

11.8 

+0.3 

5.8 

6 5 

+0.7 

2 

17.3 

18.3 

+1.0 

15.6 

16.6 

+1.0 

4 

32.9 

34.9 

+2.0 

9.3 

9.2 

-0.1 

5 

42.2 

44.1 

+1.9 

8.6 

9.2 

+0.6 

6 

50.8 

53  3 

+2.5 

10.0 

9.6 

-0.4 

7 

60.8 

62.9 

+2.1 

7.2  | 

8.0 

+0.8 

8 

68.0 

70.9 

+2.9 

8.6 

8.8 

+0.2 

9 

76.6 

79.7 

+3.1 

17.2 

21.5 

+4.3 

11 

93.8 

101.2 

+7.4 

13.0 

16.8 

+3.8 

13 

106.8 

118.0 

+11.2 

13.0 

13.5 

+0.5 

15 

119.8 

131.5 

+11.7 

22.8 

29.2 

+6.4 

18 

142.6 

160.7 

+18.1 

12.6 

12.2 

—0.4 

1 

12.6 

12.2 

—0.4 

5.6 

6.5 

+0.9 

2 

18.2 

18.7 

+0.5 

14.6 

15.5 

+0.9 

4 

32.8 

34.2 

+1.4 

9.2 

9.5 

+0.3 

5 

42.0 

43.7 

+1.7 

8.6 

9.6 

+1.0 

6 

50.6 

53.3 

+2.7 

9.5 

9.6 

+0.1 

7 

60.1 

62.9  | 

+2.8 

7.3 

7.8 

+0.5 

8 

67.4 

70.7  1 

+3.3 

8.5 

9.3 

+0.8 

9 

75.9 

80.0 

+4.1 

18.3 

21.2 

+2.9 

11 

94.2 

108.7  ! 

+7.0 

14.5 

18.2 

+3.7 

13 

101.2 

119.4  i 

+10.7 

13.5 

16.6 

+3.1 

15 

114.7 

136.0 

+13.8 

24.5 

27.5 

+3.0 

18 

139.2 

163.5 

+16.8 

Relation  of  Soil  Bacteria  to  Evaporation 


203 


The  results  here  obtained  seemingly  contradict  all  previous 
work,  for  in  the  series  receiving  manurial  additions,  the  ster- 
ile plates  exceed  the  normal  or  inoculated  ones  in  the  rate  of 
evaporation.  This  is  true  in  both  cases  where  manure  was 
added.  It  is  noteworthy,  however,  that  the  untreated  series 
showed  an  acceleration  in  the  rate  of  evaporation  in  the  inocu- 
lated plates  which  harmonizes  with  the  results  of  previous  work. 
The  cause  of  the  reduction  in  the  rate  of  evaporation  may  pos- 
sibly be  explained  by  the  chemical  composition  of  the  manure 
and  the  by-products  formed  from  it  by  bacterial  activity.  If 
these  by-products  are  of  such  a consistency  as  to  occasion  a re- 
duction in  the  surface  tension  of  the  soil  water,  then  a move- 
ment of  the  moisture  away  from  the  surface  of  the  soil  plates 
would  result.  Such  a movement  would  cause  a reduction  in 
the  amount  of  water  exposed  to  evaporation,  and  thus  would 
retard  the  rate  of  evaporation. 

The  possibility  of  error  in  the  results  of  experiments  on  the 
influence  of  the  addition  of  organic  substances,  particularly 
that  of  manure,  warranted  a repetition  of  the  same.  These 
were  performed  on  a somewhat  larger  scale,  five  series  of  plates 
being  prepared.  Both  blood  meal  and  manure  were  again  em- 
ployed using  1%,  2%,  3.5%  and  5%,  respectively,  in  contrast 
to  the  control  series  which  received  no  additions.  The  results 
of  these  experiments  are  given  in  Tables  XX  and  XXI. 

In  the  case  of  blood  meal  (Table  XX),  note  how  closely  the 
results  in  all  series  agree.  Up  to  the  ninth  day  there  is  but  little 
difference  ; thereafter,  however,  the  series  receiving  additions  of 
blood  meal  show  increased  rates  of  evaporation  due  to  inocu- 
lation, in  contrast  to  the  series  receiving  no  blood  meal.  This 
difference  is  most  marked  with  the  larger  amounts  of  blood  meal. 
While  these  results  are  not  in  direct  accord  with  those  of  the 
previous  experiment,  the  general  trend  is  none  the  less  the 
same  in  both  experiments. 

The  data  in  Table  XXI  show  a marked  uniformity  in  the  re- 
sults of  all  series  with  the  exception  of  the  series  receiv- 
ing 2 % manure.  The  difference  in  the  other  series  are  slight 
and  indicate  but  little  influence  due  to  the  manurial  additions. 
In  the  previous  experiment  the  series  receiving  manure  showed 
a decreased  rate  of  evaporation  in  the  presence  of  bacteria.  In 
the  present  experiment  the  2 % manure  series  again  shows  the 


204 


Wisconsin  Research  Bulletin  No.  23 

TABLE  XXI.  INFLUENCE  OF  ADDITION  OF  MANURE 


Loss  of  moisture 
in  grams 


Day  interval 


Sterile 


Inoc- 

ulated 


Series  I 
No  manure 


1st 

2nd 

3rd 

4th 

5th 

6th  & 7th 

8th 

9th 

10th 

11th 


19.3 

19  5 

16.5 

14.6 

16.5 

15.2 

17.2 

17.6 

19.2 

21.3 

30.0 

31.2 

15.5 

19.2 

13.0 

15.0 

17.6 

19.6 

12.2 

10.6 

Series  IT 
+1%  manure 


1st 

2nd 

3rd 

4th 

5th 

6th  & 7th 

8th 

9th 

10th 

11th 


17.6 

15.6 

17.0 

17.8 
20.3 

30.2 

15.0 

13.3 

18.0 

11.8 


18.6 

15.2 
16.6 

17.3 
20.8 
31.8 

16.3 
15.6 
19.2 
11.0 


Series  III 
+2%  manure 


1st 

2nd 

3rd 

4th 

5th 

6th  & 7th 

8th 

9th 

10th 

Uth 


18.0 

16.0 

17.2 
17.8 
22.0 

30.3 
16.5 

13.0 

18.0 
9.5 


17.0 

15.8 
16.2 

18.0 

20.6 

29.8 

17.3 

15.3 
18.0 

9.7 


Series  IV 
+3.5%  manure 


Differ- 
ence in 
1'avor  of 
inoc- 
ulated 
set 


+0.2 


—1.9 
—1.3 
+0.4 
+ 2.1 
+1.2 


+3.7 
+2.0 
+2.0  j 
—1.6 


+1.0 
-0.4 
-0.4 
—0.5 
+0.5 
+ 1.6 
+1.3 


+1.2 

-0.8 


—1.0 

—0.2 

—1.0 

+0.2 

—1.4 

—0.5 

+0.8 

+2.3 

0.0 

+0.2 


I 

Total 
num- 
ber of 
days 

Loss  of 
in  g 

Sterile 

moisture 

rams 

Inoc- 

ulated 

! Differ- 
ence in 
favor  of 
inoc- 
ulated 
set 

1 

19.3 

19.5 

+0.2 

2 

35  8 

34.1 

—1.7 

3 

52.3 

49.3 

—3.0 

4 

69.5 

66.9 

—2.6 

5 

88.7 

88.2 

—0.5 

7 

118.7 

119.4 

+0.7 

8 

134.2 

138.6 

+4.4 

9 

147.2 

153.6 

+6.4 

10 

164.8 

173.2 

+8.4 

11 

177.0 

183.8 

+6.8 

1 

17.6 

18.6 

+1.0 

2 

33.2 

33.8 

+0.6 

3 

50.2 

50.4 

+0.2 

4 

68.0 

67.7 

—0.3 

5 

88.3 

88.5 

+0.2 

7 

118.5 

120.3 

+1.8 

8 

133.5 

136.6 

+3.1 

9 

146.8 

152.2 

+5.4 

10 

164.8 

171.4 

+6.6 

11 

176.6 

183.4 

+5.8 

1 

18.0 

17.0 

—1.0 

2 

34.0 

32.8 

—1.2 

3 

51.2 

49.0 

—2.2 

4 

69.0 

67.0 

—2.0 

5 

91.0 

87.6 

-3.4 

7 

121.3 

117.4 

—3.9 

8 

137.8 

134.7 

—3.1 

9 

150.8 

150.0 

—0.8 

10 

168.8 

168.0 

—0.8 

11 

178.3 

177.7 

-0.6 

1st 

2nd 

3rd....... 

4 th 

5th 

6th  & 7th 

8th 

9th 

10th 

11th 


16.6 

17.6 

16.2 

18.5 


29.2 

14.5 

12.6 
15.8 
11.0 


17.6 
17.0 

16.6 
17.6 


32.6 

15.5 

13.8 

16.3 

10.2 


+1.0 

—0.6 

+0.4 

—0.9 

—1.2 

+3.4 

+1.0 

+1.2 

+0.5 

-0.8 


Series  V 
+5%  manure 


1 16.6 

2 34.2 

3 50.4 

4 68.9 

5 89.1 

7 118.3 

8 132.8 

9 145.4 

10  161.2 

11  172.2 


17.6 

34.6 
51.2 
68.8 
87.fi 

120.4 

135.9 

149.7 

166.0 

176.2 


+1.0 

-0.4 

+0.8 

—0.1 

—1.3 

+2.1 

+3.1 

+4.3 

+4.8 

+4.0 


1st 

2nd 

3rd 

4th 

51  h 

6th  & 7th 

8th 

9th 

10th 

11th 


19.8 

17.0 

15.3 
16.6 

19.3 

29.6 

14.3 
12.2 
15.2 

10.6 


16.2  —3.6 

16.5  —0.5 


17.0 

18.3 

21.8 

31.2 

16.2 

13.6 

16.6 
10.2 


+1.7 

+1.7 

+2.5 

+1.6 

+1.9 

+1.4 

+1.4 

—0.4 


1 19.8 

2 36.8 

3 52.1 

4 68.7 

5 88.0 

7 117.6 

8 131.9 

9 144.1 

10  159.3 

11  169.9 


16.2 

32.7 

49.7 

68.0 

89.8 
121.0 
137.2 
150.8 
167.4 
177.6 


—3.6 

-4.1 

—2.4 

-0.7 

+1.S 

+3.4 

+5.3 

+6.7 

+8.1 

+7.7 


Relation  of  Soil  Bacteria  to  Evaporation 


205 


same  decrease.  Thus  the  results  for  both  experiments  with  2 % 
manure  are  similar  and  raise  the  question  as  to  the  causal  factor 
concerned  in  producing  such  a reduction.  It  seems  strange  that 
the  addition  of  2 % manure  alone  should  exert  such  an  influence, 
whereas  other  amounts  of  manure  show  no  such  reduction.  The 
complexity  of  the  processes  involved  makes  it  difficult  to  offer  an 
explanation.  The  fact  remains  that  the  presence  of  2 % manure 
occasioned  a reduction  in  the  evaporation  due  to  inoculation. 
No  other  instance  of  such  a condition  has  been  observed  in  any 
of  the  experiments  performed. 

Problem  VI.  Influence  of  a Dry  Atmosphere  on 
Evaporation 

In  the  work  thus  far  reported  all  experiments  Avere  con- 
ducted in  the  special  room  in  which,  however,  it  was  impos- 
sible to  control  or  keep  constant  the  humidity  of  the  air.  The 
result  was  more  or  less  variation  in  the  moisture  of  the  air  re- 
sulting in  a fluctuation  in  the  rate  of  evaporation  from  day  to 
day,  being  greater  on  a bright  sunny  day  and  less  on  a dark 
rainy  day.  One  finds  as  a result,  little  uniformity  in  the  daily 
evaporations  in  the  experiments  thus  far  performed.  A series 
of  plates  was  therefore  arranged,  so  that  a constant  stream  of 
air,  dried  by  passing  through  sulfuric  acid  and  calcium  chlor- 
ide was  run  over  the  exposed  plates,  the  moisture  taken  up 
by  this  current  of  air  being  reabsorbed  by  sulfuric  acid.  The 
increase  in  weight  of  the  sulfuric  acid  bottle  was  considered  to 
indicate  the  moisture  lost  by  evaporation.  Owing  to  lack  of 
sufficient  apparatus,  only  one  plate  each  (inoculated  and  sterile) 
could  be  run  at  a time.  The  method,  however,  did  not  prove 
so  satisfactory  as  was  at  first  thought;  variations  in  the  water 
pressure  used  to  suck  the  air  through  the  apparatus,  occasioned 
marked  differences  in  the  daily  losses  due  to  evaporation.  Thus 
at.  certain  intervals  the  evaporation  per  day  was  26  grams, 
whereas  at  other  times  it  was  less  than  5 grams.  This  is  evi- 
dent from  Table  XXII.  It  is  interesting  to  note,  however,  that 
here  again  the  inoculated  set  exceeded  the  sterile  in  rate  of  evap- 
oration. After  several  futile  attempts  to  regulate  the  water 
pressure  to  give  uniform  results,  the  method  was  discarded  as 
unsuitable  for  the  work  in  hand. 


206 


Wisconsin  Research  Bulletin  No.  23 


TABLE  XXII.  EVAPORATION  IN  A DRY  ATMOSPHERE 
The  air  was  dried  by  passing  through  sulfuric  acid  and  calcium  chloride. 


Hour  interval 

Loss  of  moisture  in  grams 

Increased  evap- 
oration in  grams 
of  inoculated  set 

Sterile 

1 

I Inoculated 

1st  12 

£ 1 

0.3 

2nd  12 

D . 1 
A C 

5.4 

24t.h  to  60th '. 

i . o 

3.3 

4.7 

0.2 

60th  to  72nd 

40 

5.3 

2.0 

72nd  to  12oth 

9 1 7 

7.0 

3.0 

120th  to  1441  h.... 

9 1 

. 7 

5.0 

TAtol 

10.3 

1.2 

47.7  ~ 

59.4 

11.7 

Problem  VII.  Capillary  Action  of  the  Soil  Moisture 

It  is  evident  throughout  that  the  presence  of  bacteria  was  in- 
strumental in  accelerating  the  rate  of  evaporation.  It  seems 
hardly  probable  that  the  bacteria  themselves  are  directly  re- 
sponsible for  the  increased  evaporation  secured  when  they  were 
present,  in  fact  one  would  expect  a retardation,  if  any  influence 
at  all,  similar  to  that  secured  in  the  presence  of  the  diluted  gela- 
tin solution.  It  is  more  probable  that  the  marked  bacterial  ac- 
tivity which  undoubtedly  occurs  in  the  normal  or  inoculated 
series,  is  accompanied  by  a pronounced  metabolism  with  the 
production  of  soluble  by-products  of  an  inorganic  as  well  as  or- 
ganic nature.  These  undoubtedly  change  the  surface  tension 
of  the  soil  moisture,  whereas,  no  such  change  occurs  in  the  sterile 
controls.  The  concentration  of  the  soil  water  is  thus  modified. 
Mineral  salts  usually  increase  the  surface  tension  whereas  soluble 
organic  compounds  especially  those  of  an  oily  nature,  decrease 
it.  Whether  the  surface  tension  will  be  greater  than  that  of 
pure  water  will  depend  upon  the  proportion  of  organic  to  min- 
eral compounds  in  solution.  Thus  soil  extracts  usually  show  a 
much  lower  surface  tension  than  pure  water,  although  they  con- 
tain dissolved  salts.  The  soluble  organic  compounds  dissolved 
in  such  extracts  usually  more  than  neutralize  the  effect  of  the 
mineral  compounds,  the  result  being  a reduction  in  the  surface 
tension. 

if  during  '-he  process  of  bacterial  development,  a conversion 
of  organic  to  mineral  compounds  occurs,  then  one  should  expect 
an  increase  in  the  surface  tension.  It  is  reasonable  to  suppose 
that  the  processes  of  protein  decomposition  (ammonification  and 


Relation  of  Soil  Bacteria  to  Evaporation 


207 


nitrification)  occur  under  conditions  such  as  prevailed  in  the  ex- 
periments, as  well  as  that  an  increase  in  the  concentration  of  the 
mineral  constituents  in  the  soil  solution  takes  place  as  a result 
of  the  solvent  action  of  the  carbonated  water  produced  by  the 
bacterial  metabolism.  These  two  factors  would  tend  to  decrease 
the  amount  of  soluble  organic  matter  in  the  soil  water  and  simul- 
taneously to  increase  the  concentration  of  the  mineral  constitu- 
ents, which  process  would  mean  an  increased  surface  tension. 

Such  an  increase  in  the  surface  tension  would  mean  an  in- 
crease in  capillary  action.  If  capillary  action  is  thus  increased, 
the  tendency  would  be  to  hold  more  water  at  the  surface  of  the 
soil  as  well  as  to  bring  more  there,  than  in  the  sterile  plates 
where  no  such  increased  surface  tension  occurs.  This  exposure 
of  more  water  to  evaporation  in  the  normal  or  inoculated  series, 
would  account  for  the  difference  in  rate  of  evaporation  between 
the  inoculated  and  sterile  series  which  has  thus  far  been  ob- 
served. 

Proceeding  on  this  basis  the  following  experiment  was  ar- 
ranged to  determine  the  accuracy  of  the  above  explanation  as  to 
the  cause  of  the  increased  rate  of  evaporation. 

Percolators  were  used  because  they  were  open  at  both  ends 
thus  preventing  the  danger  of  any  possible  error  due  to  gas 
formation,  as  pressure  exerted  by  such  gas  would  be  equalized 
downward  as  well  as  upward.  The  upward  pressure  in  vessels 
closed  at  the  bottom  is  considered  in  a subsequent  experiment. 
Three  series  of  four  percolators  each  were  treated  and  arranged 
according  to  the  plan  presented  in  Table  XXIII.  In  each  se- 
ries there  were  two  normal  or  inoculated  percolators  and  two 
others  kept  sterile  by  means  of  the  weak  mercuric  chloride  solu- 
tion, being  identical  in  all  other  respects. 

The  percolators  were  allowed  to  stand  in  a warm  room  to  per- 
mit marked  bacterial  development.  The  contents  of  each  per- 
colator were  then  removed,  air-dried,  and  mixed,  separately. 
The  dried  and  well  mixed  soils  were  then  restored  to  their  re- 
spective percolators,  and  600  c.  c.  of  sterile  distilled  water  grad- 
ually added  to  the  surface  of  each.  The  excess  water  running 
through  was  retained  for  several  surface  tension  experiments 
which  will  be  discussed  later. 

After  remaining  thus  for  three  days,  moisture  determinations 
were  made  upon  the  top  and  bottom  soil  layers  in  each  of  the 


208 


Wisconsin  Research  Bulletin  No.  23 


TABLE  XXIII.  EFFECT  OF  CAPILLARITY  ON  MOISTURE  AT  SURFACE 

OF  SOIL 

exPer^m?nt  on  influence  of  increased  capillarity  upon  amount  of  moist- 
ure at  the  surface  of  soil  columns. 


Series 

Condition 

No.  of 
perco- 
lator 

Treatment 

I.  1200  grams  quartz  sand 

Normal 

1 

2 

100  c.  c.  nutrient  solution 
200  c.  c.  soil  infusion 

Sterile 

1 

2 

10®  c.  c.  nutrient  solution 
180  c.  c.  soil  infusion-j-20  c.  c. 
HgCb  solution 

II.  900  grams  clay  loam 

Normal 

2 

100  c.  c.  water 
200  c.  c.  soil  infusion 

Sterile 

1 

2 

100  c.  c.  water 

180  c.  c.  soil  infusion+20  c.  c. 
HgCb  solution 

III.  900  grams  muck 

Normal 

1 

2 

100  c.  c.  water 
200  c.  c.  soil  infusion 

Sterile 

1 

2 

100  c.  c.  water 

180  c.  c.  soil  infusion+20  c.  c. 
HgCL  solution. 

percolators.  If,  on  the  basis  of  the  explanation  given  above,  in- 
creased capillary  action  existed  in  the  inoculated  series,  then 
there  should  be  a higher  moisture  content  in  the  surface  layers 
of  the  latter  than  in  the  sterile  series.  Consultation  of  the  data 
in  Table  XXIV  reveals  such  an  increased  moisture  content  in  all 
cases,  being  greatest  in  the  muck  soil  and  least  in  the  sand. 


TABLE  XXIV.  MOISTURE  CONTENT,  BY  WEIGHT,  OF  TOP  AND  BOTTOM 
SOIL  LAYERS  IN  PERCOLATORS  CONTAINING  INOCULATED  AND 
STERILE  SOILS 


Condition 

Sand 

Per  cent  moisture 

Clay  loam 
Per  C3nt  moisture 

Muck 

Per  cent  moisture 

Top 

Bottom 

Top 

Bottom 

Top 

Bottom 

Inoculated 

5.5 

3.5 

21.8 

21.8 

30.3 

27.8 

31.0 

31.0 

37.0 

30.5 

40.0 

40.0 

Sterile 

Difference  in  favor  of  inocu- 
lated series 

2.0 

0.0 

2.5 

0.0 

6.5 

0.0 

Delation  op  Soil  Bacteria  to  Evaporation  209 

The  figures  in  Table  XXIV  are  the  average  of  the  t*wo  perco- 
lators in  each  set.  In  contrast  to  the  difference  in  the  top  lay- 
ers, one  finds  a marked  uniformity  in  the  moisture  contents  of  the 
bottom  layers  for  both  inoculated  and  sterile  series.  It  appears 
from  this  that  more  water  is  retained  in  the  top  layers  of  the 
inoculated  series,  and  that  this  is  due  to  greater  capillary  action. 
The  downward  pull  of  gravity  is  opposed  by  the  upward  force 
of  capillarity  more  in  the  inoculated  series  than  in  the  sterile 
series.  These  results  thus  apparently  substantiate  the  state- 
ment that  the  development  of  the  soil  bacteria  occasions  a change 
in  the  surface  tension  of  the  soil  water  due  to  changes  in  the 
composition  and  proportion  of  soluble  compounds  contained 
therein ; this  change  in  surface  tension  causes  greater  capillary 
action  which  means  that  more  water  is  retained  in,  and  brought 
to,  the  surface  layers  and  exposed  to  evaporation.  Why  muck 
should  exceed  the  clay  loam  and  the  sand,  can  perhaps  be  ex- 
plained on  the  basis  of  the  fineness  of  the  soil  particles.  It  is 
known  that  the  finer  the  soil  particles,  the  greater  the  number  of 
capillary  spaces,  which  in  turn  increases  the  capillary  pressure. 
Of  the  soils  used  in  the  experiments  the  muck  was  the  finest' 
grained.  No  doubt  the  greater  amount  of  insoluble  organic 
matter  in  the  muck  also  served  as  a contributing  factor  in  pro- 
ducing the  increased  difference. 

The  filtrates  obtained  from  the  percolator  experiment  were 
employed  to  determine  their  capillary  rise  in  sand  tubes  of  ordi- 
nary glass  tubing  1.2  cm.  in  diameter  and  500  cm.  long.  Each 
tube  was  sealed  at  the  lower  end  with  a cheese  cloth  cap,  and 
filled  with  quartz  sand  which  had  been  passed  through  an  80- 
mesh  sieve.  The  tubes  were  then  set  in  beakers  containing  the 
various  filtrates  and  the  rates  at  which  the  latter  ascended  by 
capillarity  in  the  sand  columns  noted.  Filtrates  from  the  inocu- 
lated soils  rose  faster  than  those  from  the  sterile  soils,  which  fur- 
ther substantiates  the  explanation  to  account  for  the  increased 
evaporation  in  the  presence  of  bacteria : namely  that  they  cause 
an  increased  capillarity. 

Problem  VIII.  Influence  of  Gas  Formation  on  Evaporation 

of  Moisture 

The  preceding  experiments  have  shown  that  there  is  a greater 
capillary  rise  of  soil  water  in  all  cases  where  bacterial  develop- 


210 


Wisconsin  Research  Bulletin  No.  23 


ment  occurs.  Evidently  in  the  process  of  bacterial  multiplica- 
tion, by-products  are  produced  which  increase  the  surface  ten- 
sion oi-  the  soil  water.  This  occasions  a greater  capillary  action 
resulting  in  a movement  of  the  soil  water  to  the  surface,  where 
it  is  exposed  to  evaporation.  Whether  this  is  the  only  factor 
contributing  as  a cause  of  the  increased  evaporation  where  bac- 
terial multiplication  occurs,  is  not  known  but  there  are  probably 
other  contributory  agencies. 

It  is  a well-known  fact  that  under  certain  conditions,  gas 
forms  in  soil.  This  is  particularly  true  where  there  are  large 
quantities  of  decaying  organic  matter  in  the  presence  of  exces- 
sive moisture,  as  under  waterlogged  conditions  in  swamps  and 
marshes.  Here  one  frequently  has  marked  evolution  of  methane. 
The  production  oP  such  gases  in  the  lower  soil  layers  is  un- 
doubtedly instrumental  in  bringing  the  moisture  from  the  lower 
soil  layers  to  the  surface,  especially  where  a condition  of  satura- 
tion prevails.  While  the  soil  water  has  a continuous  unbroken 
passage  to  the  surface  by  reason  of  its  capillary  film,  the  gas 
generated  has  not.  The  result  is,  that  the  gas  in  endeavoring  to 
escape  from  the  lower  soil  layers,  must  force  the  soil  water  in 
advance  of  it,  and  thus  brings  the  water  to  the  surface.  That 
this  phenomenon  can  occur,  was  well  demonstrated  in  the  fol- 
lowing manner.  Several  large  glass  cylinders  were  filled  with 
quartz  sand  to  a depth  of  14  inches.  To  these  were  then  added 
enough  dilute  1%,  dextrose  bouillon  to  saturate  the  sand  col- 
umns. Pour  cylinders  each  received  500  c,  c.  of  the  bouillon  and 
100  c.  c.  of  a soil  infusion.  Pour  others  each  received  a similar 
amount  of  the  bouillon  and  100  c.  c.  of  a soil  infusion  containing 
mercuric  chloride.  As  the  same  amount  of  sand  had  been  em- 
ployed in  all  cylinders,  all  had  received  identical  treatment  ex- 
cept the  addition  of  the  mercuric  chloride  to  one  set  of  four. 
This  set,  of  course,  remained  sterile. 

After  preparation  all  cjdinders  were  incubated  and  the 
changes  taking  place,  observed.  After  24  hours  the  inoculated 
set  showed  signs  of  gas  formation  and  a decidedly  moist  surface 
iii  contrast  to  the  sterile  set  which  revealed  no  gas  formation, 
and  which  were  practically  dry  at  the  surface.  After  36  hours 
there  was  an  accumulation  of  water  on  the  surface  of  the  inocu- 
lated set,  brought  there  by  the  gas  in  the  lower  layers  endeavor- 
ing to  escape  upward.  The  appearance  of  the  cylinders  at  the 


Relation  of  Soil  Bacteria  to  Evaporation 


211 


end  of  48  hours  is  shown  in  Fig.  1.  Whereas  the  surface  of  the 
sterile  set  was  perfectly  dry,  that  of  the  inoculated  was  covered 
with  1 to  2 inches  of  water,  although  both  sets  possessed  the 
same  initial  moisture  content. 


FIGURE  1.  WATER  FORCED  TO  SURFACE  BY  GAS 

Influence  of  gas  formation  in  forcing  soil  moisture  to  surface.  Arrow  points 
show  surface  of  water. 

Cylinders  1,  2,  3,  and  4 were  inoculated.  Note  water  above  sand. 

Cylinders  5,  6,  7,  and  8 were  kept  sterile.  Note  that  surface  of  sand  is  dry. 

Problem  IX.  Influence  of  Pure  Cultures  Upon  Evaporation 

Supplementary  to  the  preceding  work,  all  of  which  was  per- 
formed with  a mixed  soil  bacterial  flora,  several  experiments  were 
conducted  employing  pure  cultures  of  various  organisms  to  as- 
certain whether  the  same  increased  evaporation  could  be  secured 
under  such  conditions. 

For  this  purpose  two  experiments  were  performed,  one  with 
cultures  of  azotobacter  and  the  other  with  Bacillus  subtilis. 
The  general  technique  used  in  the  preparation  of  the  plates  was 
the  same  as  under  the  normal  soil  bacterial  flora  work,  with  the 
one  exception  that  all  the  soils  and  sand  were  sterilized  before 
inoculation.  The  substratum  material,  whether  sand  or  soil, 
was  placed  in  the  plates  in  the  desired  quantities  and  sterilized 
in  a dry  condition  in  the  autoclave.  Suspensions  of  the  organ- 


212 


Wisconsin  Research  Bulletin  No.  23 


isms  were  made  in  sterile  distilled  water,  being  made  as  uniform 
as  possible  by  vigorous  shaking  with  sand.  After  settling,  the 
supernatant  suspension  was  removed  in  two  portions.  To  one 
of  these  mercuric  chloride  solution  was  added;  to  the  other  an 
equal  amount  of  sterile  distilled  water.  The  suspensions  thus 
obtained,  the  one  sterile,  the  other  containing  living  bacteria, 


TABLE  XXV.  INFLUENCE  OF  INOCULATION  WITH  Bacillus 
Suhtilis  ON  EVAPORATION 

Clay  loam  soil,  garden  soil,  and  quartz  sand  were  used  as  substrata. 


Day  interval 

Loss  of  moisture 
in.  grants 

. Differ- 
ence 
in  favor 

Total 

num- 

Loss of  moisture 
in  grams 

> Differ- 
ence in 
favor  of 

Sterile 

Inoc- 

ulated 

of  inoc- 
ulated 
set 

ber  of 
days 

Sterile 

Inoc- 

ulated 

inoc- 

ulated 

set 

Clay  loam  soil 
1st 

28.72 

13.45 

18.35 

15.50 

15.70 

14.40 

12.55 

12.42 

24.95 

o 77 

■ 

1 

28.72 

42.10 

60.45 

76.00 

91.77 

106.17 

118.75 

131.15 

24.95 

2nd 

14*77 

— O.  ( ( 
-J-1  QO 

—3.77 

3rd 

21  20 

Tl  .06 

-4-9  QK 

2 

39.70 

—2  40 

4th 

17  70 

4-0  OA 

3 

60.90 

+0.45 

5th 

J i . 1 u 
10  QO 

4-1  OA 

4 

78.65 

+2.65 

6th 

iu . yu 

k os: 

r 1 ./CU 
4_a  ok. 

5 

95.55 

+3.78 

7th 

ID . OD 
I1?  77 

4-1  oo 

6 

110.90 

+4.77 

8th 

10.11 
13  iQ 

4-0 

7 

8 

124.60 

+5.85 

n^U.DO 

137.70 

+6.55 

White  quartz  sand 
1st 

29.70 
14.65 
18.25 
15.47 
18.55 
17.10 
15.60 

14.70 

• 

90  77 

0 QQ 

1 

29.70 

44.35 

62.60 

78.07 

96.62 

113.72 

129.32 

144.02 

26.77 

- 

2nd 

| . t ( 

10  QO 



4-1  11*7 

—2.93 

3rd 

Iu  . 06 

90  17 

r 1 .Of 
4- 1 QO 

2 

43.09 

—1.26 

4th 

10  77 

J 1 QA 

3 

63.26 

+0.66 

5 th 

JO.  ( l 

18.40 

17  CK 

4^1 .oU 

O IK 

4 

80.03 

+2.06 

6th 

— U . JD 

1 A KK 

5 

6 

98.43 

+1.81 

7th 

1 « . oo 

17.77 

15.65 

4“U.DD 
4-0  17 

116.08 

+2.38 

8th 

\ 6.  I ( 

4_  A QK 

7 

133.85 

+4.53 

-t-u.yo 

8 

149.50 

+4.48 

Garden  soil 

1st  & 2nd 

3rd 

3rd  to  5th 

5th  to  7 th 

7th  to  9th 

31.8 

17.0 

26.1 
37.3 
28.7 

30.5 
21.1 
32.1 
34.9 

30.6 

—1.3 

+4.1 

+6.0 

-2.4 

+1.9 

2 

3 

5 

7 

9 

31.8 

48.8 

74.9 
112.2 
140.9 

30.5 

51.6 

83.7 
118.6 
149.2 

—1.3 

+2.8 

+8.8 

+6.4 

+8.3 

were  then  employed  to  moisten  the  sterile  sand  or  soil  in  the 
plates.  The  same  amounts  of  liquid  were  added  to  all  plates. 
Thus  all  received  identical  treatment  with  the  one  exception  of 
the  addition  of  mercuric  chloride  to  the  sterile  sets. 

Weighings  were  made  as  usual,  the  losses  in  weight  from  day 
to  day  being  recorded  as  due  to  evaporation.  Several  soils  and 
quartz  sand  were  employed  as  substrata.  The  results  of  these 
experiments  are  recorded  in  Tables  XXV  and  XXVI. 


Relation  of  Soil  Bacteria  to  Evaporation  213 

One  finds  here  under  pure  culture  conditions  the  same  rela- 
tionship existing  between  sterile  and  inoculated  series  as  with 
the  mixed  bacterial  inoculation  of  the  earlier  experiments.  In 
all  cases  there  is  an  increased  rate  of  evaporation  in  the  inocu- 
lated series.  This  increase  was  greater  in  the  case  of  azotobac- 
ter  than  where  Bacillus  subtilis  was  employed,  which  is  strange, 

table  xxvi.  influence  of  inoculation  with  azotobacter  on 

EVAPORATION 

Garden  soil,  field  soil,  and  quartz  sand  were  used  as  substrata. 


Loss  of  moisture 

Differ- 

| 

Loss  of  moisture 

Differ- 

in grams 

ence  in 

Total 

in  grams 

ence  in 

favor  of 

num- 

favor  of 

Day  interval 

inocu- 

1  her  of 

inocu- 

Inocu- 

lated 

days 

Inocu- 

lated 

Sterile 

lated 

set 

I 

Sterile 

lated 

set 

Garden  soil 


1st  & 2nd 

31.8 

37.9 

4-6.1 

2 

31.8 

37.9 

+ 6.1 

3rd 

17.0 

21.8 

4-  4.8 

3 

48.8 

59.7 

+10.9 

3rd  to  5th 

26.1 

36.2 

+ 10.1 

5 

74.9 

96.0 

+21.1 

5th  to  7th 

37.3 

38.5 

+ 1.2 

7 

112.2 

134.5 

+22.3 

7th  to  9th 

Field  soil 

28.7 

31.5 

+ 2.8 

9 

140.9 

166.1 

+25.2 

1st 

19.5 

20.2 

+ 0.7 

1 

19.5 

20.2 

+ 0.7 

2nd 

19.4 

20.7 

+ 1.3 

2 

38.9 

40.9 

+ 2.0 

3rd 

16.3 

19.8 

+ 3.5 

3 

55.2 

60.7 

+ 5.5 

4th 

15.5 

19.0 

+ 3.5 

4 

70.7 

79.7 

+ 9.0 

5th 

18.3 

20.2 

+ 1.9 

5 

89.0 

99.9 

+ 10.9 

6th 

17.9 

20.4 

+ 2.5 

6 

106.9 

120.3 

+13.4 

7th 

18.4 

23.0 

+ 4.6 

7 

125.3 

143.3 

+18.0 

8th  

19.1 

21.5 

+ 2.4 

8 

144.4 

164.8 

+20.4 

9th 

15.2 

17.9 

+ 2.7 

9 

159.6 

182.7 

+23.1 

10th 

13.3 

16.1 

+ 2.8 

10 

172.9 

198.8 

+25.9 

11th t, 

11.5 

12.3 

+ 0.8 

11 

184.4 

211.1 

+26.7 

12th 

8.0 

7.9 

— 0.1 

12 

192.4 

219.0 

+26.6 

13th 

Quartz  sand 

7.8 

7.5 

— 0.3 

13 

200.2 

226.5 

+26.3 

1st 

22.0 

22.3 

+ 0.3 

1 

| 22.0 

22.3 

f + 0.3 

2nd 

20.9 

21.8 

+ 0.9 

2 

42.9 

44.1 

+ 1.2 

3rd 

18.1 

19.6 

+ 1.5 

3 

61.0 

63.7 

+ 2.7 

4th 

18.6 

20.3 

+ 1.7 

4 

79.6 

84.0 

+ 4.4 

5th 

19.9 

21.8 

+ 1.9 

5 

99.5 

105.8 

+ 6.3 

6th  

19.7 

23.1 

+ 3.4 

6 

119.2 

128.9 

+ 9.7 

7th 

23.5 

25.8 

+ 2.3 

7 

142.7 

154.7 

+12.0 

8th 

21.0 

24.0 

+ 3.0 

8 

163.7 

178.7 

+ 15.0 

9th 

16.0 

20.3 

+ 4.3 

9 

179.7 

199.0 

+19.3 

10th 

11.7 

15.3 

+ 3.6 

10 

191.4 

214  3 

+22.9 

11th 

8.5 

9.5 

+ 1.0 

11 

199.9 

223.8 

+23.9 

12th  

5.1 

2.8 

— 2.0 

12 

205.0 

226.6 

+21.6 

13th 

3.8 

0.5 

— 3.3 

13 

208.8 

227. 1 

+18.3 

as  with  azotobacter  one  has  the  production  of  a more  or  less 
slimy  zoogloeal  mass  which  on  drying  assumes  a tough  gelatin- 
ous consistency  in  which  form  one  would  expect  it  to  hinder 
evaporation.  Instead,  one  finds  a very  marked  acceleration, 
probably  due  to  the  vigorous  growth  which  azotobacter  made 
under  the  conditions  of  the  experiments.  The  work  on  the  pure 
cultures  thus  further  substantiates  the  previous  work  and 


214 


Wisconsin  Research  Bulletin  No.  23 


strengthens  the  conclusion  drawn,  namely  that  the  presence  of 
bacteria  increases  the  rate  of  evaporation  from  the  soil. 

Summary 

Any  conclusions  which  can  be  drawn  from  this  work  must  be 
considered  with  great  care  to  avoid  any  erroneous  deductions  or 
applications.  It  must  be  remembered  that  the  conditions  pre- 
vailing in  the  experiments  only  approximate  field  conditions.  It 
is  believed  that  the  phenomena  here  observed  do  exert  an  in- 
fluence under  field  conditions.  The  movement  of  soil  water  is 
known  to  be  dependent  upon  such  forces  as  gravitation,  surface 
tension,  capillarity,  temperature,  and  viscosity,  as  well  as  upon 
the  chemical  and  physical  composition  of  the  soil  itself,  but  to 
these  must  be  added  the  biological  activities  taking  place  in  the 
soil. 

Changes  in  the  quantity  and  quality  of  the  various  chemical 
substances  in  solution  in  the  soil  water  result  from  bacterial 
activities.  These  changes  probably  disturb  the  equilibrium  of 
the  soil  moisture  so  far  as  its  distribution  is  concerned,  and  help 
to  cause  its  movement  and  circulation. 

The  principal  effect  of  the  bacterial  activities  is  undoubtedly 
upon  the  surface  tension  of  the  soil  moisture.  Any  change  pro- 
duced in  this,  means  a diffusion  and  a movement  of  the  soil  wa- 
ter from  the  point  of  lower  tension  to  that  of  higher  tension,  in 
an  endeavor  to  readjust  itself  uniformly  throughout'  the  soil 
mass ; in  other  words  capillary  action  will  come  into  play.  Thus, 
if  through  the  addition  of  soluble  mineral  salts,  artificially  or  as 
a result  of  the  conversion  of  insoluble  substances  to  soluble  com- 
pounds in  the  process  of  protein  decomposition  and  mineraliza- 
tion, the  surface  tension  of  the  water  in  the  upper  layers  is  in- 
creased there  will  result  an  upward  movement  to  that  point. 
This  is  probably  what  occurred  in  the  percolator  experiment 
where  the  upper  soil  layers  of  the  inoculated  series  showed  a 
higher  moisture  content  than  the  corresponding  soil  layers  in 
the  sterile  series. 

Again  if  there  are  substances  present  which  possess  the  prop- 
erty of  occasioning  a reduction  in  surface  tension  such  as  alcohol 
produces  in  water,  and  which  are  formed  as  by-products  in  the 
decomposition  of  certain  protein  or  carbonaceous  materials,  a 
reduction  in  surface  tension  occurs,  and  a movement'  away  from 


Relation  of  Soil  Bacteria  to  Evaporation 


215 


the  surface  results.  It  is  possible  that  such  a condition  occurred 
in  the  manure  experiment,  the  one  instance  where  the  inoculated 
series  showed  a less  rapid  rate  of  evaporation  than  the  sterile 
series. 

The  production  of  carbon  dioxide  by  bacteria  in  soil  is  an- 
other factor  which  undoubtedly  increases  the'  surface  tension. 
This  may  be  considerable,  amounting  in  some  instances  as  Stok- 
lasa4  has  shown,  to  1.5  liters  per  kilogram  of  soil  per  year.  The 
carbon  dioxide  thus  formed  is  dissolved  in  the  soil  water,  pro- 
ducing an  acid  reaction,  increasing  thereby  more  or  less  mark- 
edly the  solvent  action  of  the  soil  water  upon  inorganic  mineral 
compounds  with  which  it  comes  in  contact.  The  increased  solu- 
tion of  such  mineral  substances  undoubtedly  increases  the  sur- 
face tension  and  results  in  a movement  of  the  soil  water. 

The  production  of  gaseous  compounds  in  the  lower  soil  layers 
is  not  a frequent  occurrence,  as  the  conditions  necessary  for  such 
are  found  only  in  low,  swampy  places,  marshes,  etc.,  where  a 
waterlogged  condition  is  combined  with  the  presence  of  large 
amounts  of  organic  matter.  Here  gas  production  such  as  oc- 
curred in  the  experiment  with  the  glass  cylinders  may  produce 
a similar  condition  and  tend  to  prevent  the  percolation 
water  through  the  soil,  keeping  it  on  the  surface.  One  would 
not  expect  gas  formation  under  other  natural  conditions  and 
accordingly  little  stress  can  be  laid  upon  the  factor  of  gas  forma- 
tion as  an  agent  in  causing  the  increased  evaporation  observed. 

In  general  then,  it  would  appear  from  the  experiments  that 
the  soil  bacteria  and  their  activities  are  factors  which  must  he 
considered  when  discussing  the  movement  of  soil  water;  not  so 
much  because  of  the  cells  themselves  as  because  of  the  by-prod- 
ucts which  they  form  and  the  subsequent  influence  of  the  same 
upon  such  factors  as  surface  tension,  capillarity,  viscosity,  etc.  of 
the  soil  moisture.  The  biological  feature  of  the  soil  apparently 
forms  an  important  contributory  factor  in  determining  the  move- 
ment of  soil  water. 


4 Centbl.  Bakt.,  Abt.  II,  29,  1911,  p.  409. 


* 


The  Diagnosis  of  Contagious  Abortion  in 
Cattle  by  Means  of  the  Complement 
Fixation  Test 


F.  B.  HADLEY  and  B.  A.  BEACH 

The  importance  of  contagious  abortion  from  the  economic 
viewpoint  can  not  be  overestimated.  The  losses  incident  thereto 
usually  extend  over  a number  of  years  with  each  infected  ani- 
mal. Not  only  is  the  calf  lost,  due  to  its  premature  expulsion 
from  the  womb,  but  the  milk  yield  of  the  cow  is  materially  re- 
duced; she  frequently  fails  to  conceive,  and  her  productivity 
thus  becomes  impaired  for  varying  lengths  of  time. 

The  disease  exists  in  nearly  all  sections  of  the  wmrld  where 
dairying  is  engaged  in  to  any  extent.  It  is  more  prevalent  in 
northern  portions  of  the  United  States,  on  account  of  the 
greater  development  of  the  dairy  industry,  than  in  the  southern 
parts.  Well  informed  men  maintain  that  very  few  breeders 
who  have  been  in  the  business  six  or  eight  years  can  truthfully 
say  they  have  not  experienced  trouble  with  the  disease.  For 
these  reasons,  live  stock  producers  should  be  interested  in  the 
different  aspects  of  this  highly  important  and  extensively  dis- 
tributed disease. 

Contagious  abortion  is  most  frequently  seen  in  the  bovine 
species,  and  is  caused  by  a specific  microorganism  which  finds 
the  pregnant  uterus  a particularly  favorable  location  for  growth. 
It  is  usually  characterized  by  the  expulsion  of  the  fetus  before 
the  period  of  gestation  has  been  completed. 

Historical  References 

Until  a few  years  ago  it  was  thought  that  contagious  abortion 
was  caused  by  a number  of  different  organisms;  that  non©  of 
them  wer©  spepific  to  the  exclusion  of  the  others 


218 


Wisconsin  Research  Bulletin  No.  24 


The  Scottish  Commission1  found  as  many  as  five  different 
organisms  in  the  genital  tract  of  aborting  cows.  In  France, 
Nocard2  found  coccus-like  and  very  delicate  rod-shaped  organ- 
isms which  lived  in  the  womb  throughout  the  interval  between 
pregnancies. 

By  far  the  most  important  of  all  the  contributions  to  this  sub- 
ject is  that  of  Professor  Bang3  who  in  1895  undertook  the  in- 
vestigation of  this  disease.  He  found  many  very  small  bacilli 
in  stained  preparations  of  the  uterine  exudate  between  the  fetal 
membranes  and  the  wall  of  the  uterus  of  a cow  which  had  shown 
signs  of  abortion.  This  investigator  was  able  to  cultivate  this 
organism  in  pure  culture  on  gelatin-agar-serum,  and  also  to  pro- 
duce abortion  experimentally  and  recover  the  specific  organism. 

Nowak4  in  Austria  made  gelatin-agar-serum  plates,  allowed 
them  to  solidify,  after  which  they  were  streaked  with  material 
from  a suspected  subject  and  placed  in  a sealed  jar  containing 
culture  of  Bacillus  subtilis  to  exhaust  the  oxygen,  one  square 
inch  of  culture  surface  to  every  15  c.  c.  jar  capacitjL 

In  this  country  Dr.  AY.  J.  MacNeal,5 6  formerly  of  the  Illinois 
Experiment  Station,  Prof.  E.  S.  Good0  of  the  Kentucky  Experi- 
ment Station,  and  other  investigators  have  succeeded  in  isolat- 
ing an  organism  which  the}^  believe  to  be  the  Bang  bacillus. 

Etiology 

The  causal  agent  in  contagious  abortion  of  cows  has  been 
named  the  Bacillus  abortus  (Bang).  It  is  a cocco-bacillus  0.8 
to  2 microns  long  by  0.5  to  0.7  wide.  The  organism  stains  with 
the  aniline  dyes  and  is  Gram  negative.  AYhen  carbol  fuchsin  is 
used,  it  appears  much  larger  than  when  stained  with  methylene 
blue  or  gentian  violet.  Microscopical  examination  will  reveal  a 
very  small  coccus-like  organism  which  may  easily  be  mistaken 
for  the  ordinary  pus  cocci.  Close  scrutiny,  however,  will  show 

1 Woodhead,  McFadyean  & Aitkin.  Report  of  the  Scottish  Commis- 
sion. 

2 Nocard,  Review  by  Bang.  Zeitschrift  fur  Thiermed.  I.  1897. 

3 Bang.  Die  Aetiologie  des  seuchenhaften  (“infectiosen”)  Verwer- 
tens.  Zeitschrift  fur  Thiermed.  I.  1897. 

4 Nowak.  Le  bacille  de  Bang  et  sa  biologie.  Annales  de  l’lnstitut 

Pasteur.  Vol.  22;  pp.  541-546.  1908. 

s McNeal,  W,  J.  and  Mumford,  H.  W.,  111.  Bui.  152.  1911. 

6 Good,  E.  S.  The  Etiology  of  Contagious  Abortion.  Am.  Vet.  Rev. 
Vol,  XL,  No.  4.  Jan.  1912. 


Complement  Fixation  Test  for  Contagious  Abortion  219 


that  some  of  the  organisms  have  a slightly  elongated  appearance, 
the  more  striking  if  some  micrococcus  is  observed  at  the  same 
time. 

Blood-serum-agar  or  bouillon  has  heen  found  the  best  medium 
for  the  isolation  and  growth  of  the  abortion  bacillus.  (See  Fig- 
ures 1 and  2).  It  is  usually  necessary  to  grow  a strain  recently 
isolated  in  a rarefied  atmosphere  or  in  a tension  of  oxygen  some- 
what greater  than  one  atmosphere.  Growth  under  these  condi- 
tions takes  three  to  four  days.  The  behavior  towards  oxygen 
and  the  fact  that  growth  is  slight  or  does  not  occur  at  all  on 
ordinary  culture  media  are  valuable  means  of  identification. 


FIGURE  1.  HORSE  SERUM-AGAR  FIGURE  2.  OX  SERUM-AGAR 


Serum-agar  plates  of  Bacillus  abortus  after  seventy-two  hours  incubation. 
I he  inoculations  were  made  directly  from  the  stomach  contents  of  an  aborted 
fetal  calf.  Note  the  larger,  more  luxuriant  growth  on  the  ox  serum-agar. 

After  a strain  lias  been  transferred  several  times,  it  will  usually 
grow  fairly  well  under  atmospheric  conditions  on  blood-serum 
media.  Some  strains  seem  to  acquire  the  faculty  of  growing  on 
ordinary  media ; others  do  not.  One  strain  which  was  isolated 
grew'  under  ordinary  atmospheric  conditions  immediately.  It 
would  also  grow  slightly  on  plain  agar.  The  colonies  are  round, 
slightly  convex,  smooth,  and  translucent ; simulating  a honey-  or 
dew-drop.  When  observed  by  transmitted  light  a characteristic 
bluish  cast  is  noticeable. 

Bang  and  Stribolt  demonstrated  that  there  were  two  optima 
as  regards  oxygen  tension.  With  the  gelatin-agar-serum  in  pure 
oxygen  the  growth  developed  best  in  two  zones;  one  near  the 
surface,  the  other  near  the  bottoin  of  the  tube.  They  concluded 


220 


Wisconsin  Research  Bulletin  No.  24 


that  it  was  neither  a strict  aerobe  nor  an  anaerobe,  but  that  it 
required  a tension  of  oxygen  either  somewhat  greater  than  one 
atmosphere  or  a little  less. 

Microscopic  examination  will  often  reveal  a small  organism 
in  the  uterine  exudate  of  a cow  which  has  recently  aborted,  in 
the  scrapings  of  the  fetal  membranes,  the  umbilicus,  and  in  the 
stomach  contents  of  the  aborted  fetus.  Mohler7  calls  attention 
to  the  experiment  of  Schroeder  and  Cotton8  in  which  micro- 
organisms similar  to  the  abortion  bacilli'  were  found  in  guinea 
pigs  that  had  been  injected  with  milk  from  infected  cows. 

The  plate  method  of  isolation  is  used  altogether.  Ordinary 
plain  agar  is  heated  until  it  liquefies,  when  it  is  cooled  to  about 
50°  C.  Naturally  sterile  blood  serum  is  added,  approximately 
1 c.  c.  of  serum  to  every  5 c.  c.  of  agar.  The  tubes  are  kept  at 
the  above  temperature.  The  abdominal  cavity  of  the  fetus  is 
opened,  revealing  the  stomach,  the  wTall  of  which  is  seared  with 
a hot  iron,  after  which  it  is  incised  with  a sterile  scalpel.  A 
loopful  of  the  stomach  contents  is  at  once  transferred  to  a tube 
of  the  medium,  a dilution  tube  is  inoculated  from  the  first  and 
the  contents  of  both  are  immediately  poured  into  sterile  petri 
dishes.  When  the  medium  has  solidified  the  plates  are  placed 
in  a jar  containing  Bacillus  subtilis  after  the  method  of  Nowak. 
In  place  of  the  B.  subtilis  the  air  may  be  exhausted  to  200  mm. 
by  use  of  an  ordinary  vacuum  pump.  An  examination  is  made 
after  four  to  five  days’  incubation. 

Clinical  Features 

A bulletin  of  this  nature,  in  which  but  one  phase  of  the  con- 
tagious abortion  problem  is  considered  in  detail,  must  limit  ref- 
erence to  the  clinical  aspects  of  the  disease.  Therefore,  many 
interesting  and  important  points  relative  to  the  pathology, 
symptoms,  treatment,  etc.,  will  necessarily  be  omitted.  However, 
before  taking  up  the  diagnostic  methods  with  which  our  experi- 
ments have  been  particularly  concerned,  it  may  be  well  to  call 
attention  to  a few  facts  which  will  aid  the  reader  to  understand 
the  nature  of  the  malady. 


7 Bur.  Anim.  Indus.  Cir.  198.  1912. 

s Schroeder.  E.  C.  and  Cotton,  W.  E.  Proc.  Am.  Med-  Vet.  Assoc,  pp. 
195-206.  1911 


Complement  Fixation  Test  for  Contagious  Abortion  221 

The  principal  pathological  lesions  produced  as  a result  of  in- 
fection with  the  abortion  bacilli  occur  in  the  pregnant  uterus 
and  its  contents.  When  present  in  large  numbers  the  organ- 
isms appear  to  set'  up*  an  inflammation  in  the  fetal  membranes, 
especially  at  the  points  of  union  with  the  maternal  membranes, 
which  in  the  cow  are  known  as  cotyledons.  These  finally  be- 
come involved  to  such  an  extent  that  the  natural  exchange  of 
gases  and  nutrients  can  no  longer  take  place  between  the  mother 
and  the  fetus  with  the  result  that  abortion  or  premature  birth 
usually  occurs.  The  placental  membranes  sometimes  become  ab- 
normally thickened  and  inflamed,  due  to  the  irritation  of  the 
bacilli.  Possibly  certain  toxins,  the  products  of  bacterial  growth, 
are  influential  in  the  inflammatory  changes,  but  further  re- 
searches must  be  made  along  this  line  before  this  supposition  is 
proved.  Frequently  a peculiar  granular  or  nodular  ap- 
pearance of  the  placenta  is  to  be  noted  as  the  result  of  this  in- 
fection. Many  veterinary  practitioners  regard  this  condition 
as  pathognomonic  of  contagious  abortion. 

Infection  may  gain  entrance  to  the  body  by  a number  of  dif- 
ferent routes.  The  genital  passage  is  a frequent  avenue  for  the 
entrance  of  the  bacilli.  Experiments  made  by  the  British  Com- 
mission9 indicate  that  the  mouth  must  be  considered  an  import- 
ant portal  for  the  entry  of  these  disease  producing  organisms. 
Artificial  infection  with  natural  material  and  cultures  of  the 
abortion  bacillus  by  subcutaneous  and  intravenous  inoculations 
have  also  been  demonstrated  to  be  methods  by  which  the  cow 
could  be  caused  to  abort. 

The  discharges  from  the  uterus  of  a diseased  cow  are  prob- 
ably the  chief  source  of  infection.  Not  only  are  they  dangerous 
before  and  at  the  time  of  abortion,  but  also  for  an  indefinite 
period  thereafter.  There  is  evidence  to  support  the  belief  that 
occasional  cases  may  act  as  carriers  of  the  abortion  bacilli  for 
many  months  after  the  last  abortion,  as  do  the  so-called  typhoid 
fever  carriers  of  the  human  race. 

It  is  commonly  held  that  the  bull  may  convey  the  infection. 
Undoubtedly  a bull  may,  after  serving  an  infected  cow,  carry  on 
his  copulat'orv  organ  infectious  material  to  the  cows  he  subse- 
quently serves  so  that  they  too  become  infected.  However,  at- 

9 Report  of  Departmental  Committee  to  Inquire  into  Epizootic  Abor- 
tion. Appendix  to  Part  1.  London,  1909. 


222 


Wisconsin  Research  Bulletin  No.  24 


tempts  to  infect  heifers  in  this  way  have  usually  been  unsuccess- 
ful. 

The  symptoms  exhibited  in  the  early  months  of  pregnancy  by 
a cow  about  to  abort  are  frequently  so  slight  as  to  pass  unno- 
ticed. The  incompletely  developed  fetus  is  expelled  while  the 
cow  is  at  pastuie,  or  in  the  stable  at  night,  and  is  so  small  that 
it  may  escape  the  attention  of  the  attendant.  Aside  from  the 
slight  discharge  from  the  vagina  and  swelling  of  the'  udder,  the 
animal  may  show  no  evidences  of  abortion.  Later  in  gestation, 
a marked  enlargement  of  the  udder,  swelling  of  the  external 
genital  organs,  and  a vaginal  discharge  will  be  noted,  together 
with  the  other  symptoms  which  usually  accompany  a normal 
parturition. 

Under  ordinary  farm  conditions  abortion  occurs  from  the  sec- 
ond to  the  seventh  month  of  pregnancy.  Fetuses  expelled  later 
than  this,  if  they  live,  should  rightly  be  called  premature  births, 
but  usage  has  stretched  the  term  abortion  to  include  any  birth 
before  the  normal  term  of  gestation  is  completed. 

Our  knowledge  so  far  as  the  treatment  goes  is  very  limited ; 
most  medicinal  agents  now  employed  are  used  empirically  and 
have  no  scientific  basis.  For  these  reasons  no  attempt  will  be 
made  to  discuss  the  subject  of  therapeutics  at  this  time. 

Handling  Infected  Animals 

Because  no  effective  curative  treatment  is  at  hand  and  on  ac- 
count of  the  facility  with  which  abortion  can  now  be  diagnosed, 
methods  of  prevention  will  be  considered  more  in  detail.  The 
most  emphasis  must  be  placed  upon  the  great  importance  of 
properly  disposing  of  the  placentae,  fetuses,  and  contaminated 
litter,  especially  as  the  abortion  bacilli  have  been  found  to  re- 
tain their  vitality  for  a considerable  time  outside  the  body. 

The  following  preventive  measures  may  be  suggested:  Re- 

move and  bury  infectious  material  deeply  or  burn  it.  Disinfect 
the  gutters,  floors,  and  walls  with  a 1-1000  solution  of  corrosive 
sublimate.  Avoid  buying  cows  from  a district  where  abortion  is 
known  to  exist.  Unless  the  complement  fixation  test  can  be  ap- 
plied when  new  cows  are  purchased,  it  is  well  to  quarantine  such 
animals  until  they  calve.  The  vulva,  tail,  thighs,  and  udder  of 
each  cow  in  an  infected  herd  should  be  washed,  sponged,  or 
sprayed  with  a disinfectant  solution  each  day.  Those  cowrs 


Complement  Fixation  Test  eor  Contagious  Abortion  223 

which  abort  should  be  irrigated  with  a disinfectant  douche. 
This  douche  can  not  be  used  in  sufficient  strength  to  destroy  the 
germs  on  account  of  the  irritating  effects  upon  the  mucous  mem- 
brane, but  it  will  aid  in  the  prompt  removal  of  the  infectious  or- 
_ ganisms.  In  our  opinion,  the  irrigation  should  be  made  at  least 
once  a day  and  continued  until  all  evidence  of  the  discharge  has 
ceased.  A 0.5  to  1%  solution  of  any  reliable  coal  tar  disinfectant, 
or  a 1-2000  solution  of  potassium  permanganate  may  be  used  for 
this  purpose.  Cows  in  oestrum  are  more  susceptible  to  irritants. 
In  these  cases  a 3%  solution  of  boric  acid  or  bicarbonate  of 
soda  may  be  substituted.  Bulls  which  have  recently  served  in- 
fected cows  should  be  disinfected  by  flushing  out  the  sheath  be- 
fore another  cow  is  covered.  If  an  animal  shows  premonitory 
symptoms  of  abortion  she  should  be  immediately  removed  from 
the  herd. 

The  best  way  to  handle  cows  which  have  aborted,  also  those 
which  have  given  a positive  reaction  to  the  complement  fixation 
'test,  is  to  isolate  them  from  the  noninfected  animals.  As  already 
pointed  out,  there  is  ample  reason  to  believe  that  certain  cows 
harbor  the  infectious  organism  for  a considerable  period  after 
the  abortion,  otherwise  there  would  be  no  plausible  explanation 
for  the  recurrence  of  the  disease  in  these  animals  the  following 
year.  Such  cows  are  to  be  regarded  as  possible  sources  of  in- 
fection and  should  not  be  stabled  with  noninfected  cattle. 
Many  infected  cows  will  not  readily  conceive  after  infection,  in 
fact  some  remain  barren  permanently  and  soon  become  nonsup- 
porting. For  these  reasons  it  is  better  to  either  dispose  of 
these  animals  for  beef  purposes,  or  maintain  a separate  herd, 
stable,  and  pasture. 

The  policy  adopted  at  this  station  is  to  test  all  cattle  in  the 
herds  each  year.  Any  new  animals  proposed  to  be  introduced 
are  tested  before  they  are  placed  with  the  regular  herd.  By 
these  means,  it  is  expected  that  the  disease  will  be  prevented 
here. 

It  is  well  known,  that  a certain  degree  of  immunity  is  confer- 
red by  an  attack  of  abortion.  In  one  dairy  herd  composed  of 
700  animals,  where  careful  records  were  kept,  133  aborted  in  a 
period  of  three  years.  Of  these  aborters  which  again  conceived, 
28.6%  aborted  a second  time,  14.2%  the  third  time,  thus  leav- 


224 


Wisconsin  Research  Bulletin  No.  24 


ing  57.2%  which  aborted  but  once.  Many  of  the  last  mentioned 
must  therefore  have  acquired  au  immunity. 

Recently  the  statement  has  been  made  that  a herd  of  cows 
which  had  aborted  would  be  more  valuable  for  breeding  pur- 
poses, on  account  of  the  acquired  immunity,  than  a stock  that 
had  never  been  exposed  to  the  infection.  This  would  probably 
be  true  in  a locality  where  the  disease  was  epizootic,  but  would 
only  be  a temporary  asset,  as  a large  herd  of  this  kind  would 
undoubtedly  contain  certain  cows  which,  although  immune  them- 
selves, would  harbor  the  disease  producing  organisms.  Such 
cows  would  be  capable  of  transmitting  the  infection  not  only  to 
aH  newly  purchased  cattle,  but  also  to  the  calves  from  the  im- 
mune cows  which,  unfortunately,  are  not  always  rendered  con- 
genitally immune.  Besides,  a breeder  who  makes  a practice  of 
raising  pure  bred  stock  for  breeding  purposes  would  soon  gain 
a bad  reputation  if  he  were  known  to  maintain  such  a herd. 


Methods  of  Diagnosing  Contagious  Abortion 

Among  the  various  methods  devised  for  the  diagnosis  of  con- 
tagious abortion  may  be  mentioned:  the  agglutination  test;  the 
reaction  method;  a bacteriological  examination;  and  the  comple- 
ment fixation  test. 

The.  Agglutination  Test.  This  test  has  been  used  by  various 
investigators  but  by  itself  has  not  been  found  uniformly  satis- 
factory. One  reason  for  this  is  that  an  increase  in  the  agglutina- 
ting power  of  sera  from  cows  which  have  recently  become  in- 
fected does  not  occur  for  some  time.  Experiments  seem  to  show 
that'  a diagnosis  based  upon  the  agglutination  method  alone,  is 
only  fairly  reliable  at  best,  but  in  a combination  with  other 
methods  it  will  be  found  serviceable. 

The  EeacUon  Method.  This  method  is  based  upon  the  suc- 
cess attained  by  the  use  of  tuberculin  and  mallein,  and  consists 
of  the  subcutaneous  injection  of  an  agent'  prepared  in  a manner 
similar  to  that  used  in  the  manufacture  of  these  diagnostic 
agents.  McFadyean  and  Stockman  have  called  this  product 
abortin.  ’ ’10  Their  experiments  with  it  were  confined  to  a rel- 
atively small  number  of  animals.  The  results  were  not  satis- 
factory enough  to  warrant  any  definite  statement  as  to  its  value. 


Report  of  Departmental  Committee}  to  Inquire  into  Epizootic  Abor- 
tion. Appendix  to  l art  I.  London,  1909. 


Complement  Fixation  Test  for  Contagious  Abortion  225 


Bacteriological  Examination.  This  method  of  diagnosis  in- 
volves a microscopical  and  cultural  examination  of  the  placental 
membranes,  the  fetus,  and  the  uterine  exudate.  Stained  slide 
preparations  are  made  from  scrapings  taken  from  the  coty- 
ledons, and  other  parts  of  the  placenta,  the  stomach  fluid  of  the 
fetus,  and  from  the  uterine  exudate.  When  the  abortion  ba- 
cilli are  present  in  large  numbers,  they  may  be  demonstrated 
in  such  preparations.  The  most  satisfactory  results  are  ob- 
tained, however,  by  making  cultural  examinations  of  fresh  ma- 
terial on  serum-agar  medium.  The  characteristic  honey-  or 
dew-drop-like  colonies  will  appear  after  three  to  five  days  incu- 
bation under  the  proper  oxygen  tension. 

The  Complement  Fixation  Test.  Certain  infectious  diseases, 
such  as  syphilis  and  glanders  have  recently  been  brought  under 
better  control  by  this  remarkable  test  which  was  introduced  to 
the  attention  of  scientists  by  Bordet  and  Gengou.  It  is  based 
upon  the  hemolytic  action  of  a specially  prepared  blood  serum 
used  in  connection  with  blood  sera  of  other  species  of  animals. 

Use  has  lately  been  made  of  this  test  in  the  diagnosis  of  con- 
tagious abortion  by  a number  of  European  investigators,  par- 
ticularly Holth11  and  Wall,12  but  so  far  as  we  know  nothing  has 
yet  been  published  as  to  any  extensive  work  done  in  this  country. 

To  demonstrate  the  practical  application  of  the  test,  we  have 
carried  out  more  than  500  examinations  on  sera  from  animals  in 
a number  of  widely  separated  herds,  which  are  kept  under  vari- 
ous conditions  and  may  therefore  be  taken  as  fairly  representa- 
tive of  the  Wisconsin  dairy  industry. 

The  test  is  in  reality  quite  complicated  and  can  be  performed 
only  in  a properly  equipped  laboratory.  However,  a skilled 
technician  can  easily  test  50  or  more  animals  a day.  No  more 
time  is  therefore  consumed  than  in  testing  a like  number  of 
cattle  for  tuberculosis. 

For  complete  .details  concerning  the  theories  involved  in  the 
interesting  phenomenon  of  hemolysis,  which  underlies  the  foun- 
dation of  the  test,  refer  to  some  of  the  original  articles  by  Ehr- 

11  Holth,  H.  Unterschungen  fiber  die  Biologie  des  Abortusbazillus  und 
die  Immunitatsverhaltnisse  des  infectiosen  Abortus  der  Rinder.  Zeits- 
chrift  fiir  Infectionskrankheiten  der  Haustiere.  Band  10,  Heft  4/5 
1911. 

12  Wall,  S.  fiber  die  Feststellung  des  seuchenhaften  Abortus  beim 
Rinde  durch  Agglutination  and  Komplementbindung.  Zeitschrift  fiir 
Infectionskrankheiten  der  Haustiere.  Rand  10,  Heft  1/2/3.  1911. 


226 


Wisconsin  Research  Bulletin  No.  24 


lich,  Morgenroth,  Sachs13  and  others.  Only  such  fundamental 
points  will  be  here  considered  as  will  enable  one  to  understand 
the  relationship  of  the  different  components  used  in  the  comple- 
ment fixation  test  as  applied  to  contagious  abortion. 


Components  Used  in  the  Complement  Fixation  Test 


Five  separate  and  distinct  factors  with  a definite  relationship 
to  one  another  are  required,  as  follows:  (1)  the  suspect’s  blood 
serum;  (2)  antigen  or  prepared  cultures  of  the  abortion  bacil- 
lus; (3)  complement  or  fresh  guinea  pig  blood  serum;  (4)  hemo- 
lysin or  blood  serum  from  a specially  treated  rabbit;  (5)  washed 
red  horse  blood  'Corpuscles. 

When  these  substances  are  mixed  in  measured  quantities  and 
under  proper  conditions  they  may  unite  in  different  ways. 
One  possible  combination  is  the  fixation  of  the  complement  by 
the  antigen  and  antibody ; another  is  a union  of  the  complement 
and  corpuscles  by  means  -of  the  hemolysin,  resulting  in  hem- 
olysis. 

It  is  readily  seen  that  the  components  must  be  used  in  definite 
quantities,  for  example,  if  an  excess  of  the  complement  is  used 
in  a certain  test  in  which  the  antigen  and  antibody  are  present 
in  proper  amounts,  a portion  is  left  free  to  act  with  the  hemoly- 
sin and  corpuscles  to  bring  about  hemolysis — an  erroneous  re- 
sult. 

The  reaction  may  be  graphically  illustrated  in  the  case  of  a 
positive  serum,  containing  the  specific  antibodies,  as  follows: 


(After  first  Incubation,  complement  is  fixed) 


Antigen  -f-  Antibody  Complement 


Antigen 

Antibody 

Complement 


Antigen 

Antibody 

Complement 


4-  Hemolysin  + Corpuscles  .==■ 


(After 


further  incubation,  no  hemolysis) 


Antigen 

Antibody 

Complement 


4-  Hemolysin  -j- 


Corpuscles 


13  Ehrlich,  Paul.  Studies  in  Immunity.  New  York,  1910. 


Complement  Fixation  Test  for  Contagious  Abortion  227 


No  hemolysis  takes  place  here  because  the  complement  has  been 
fixed  or  bound  at  the  first  incubation  and  so  is  not  available  to 
act  with  the  hemolysin  on  the  corpuscles. 

In  the  case  of  a negative  reaction  where  no  antibodies  exist  in 
the  serum  the  results  would  be  as  follows : 


("After  first  incubation,  no  fixation) 

Antigen  4-  Serum  + Complement  ==  Antigen  -f-  Serum  4- 
Complement 

Antigen  + Serum  + Complement  + Hemolysin  + Corpus- 


ft 


cles  = 


(After  further  incubation,  hemolysis) 


Antigen  -f-  Serum 


Corpuscles 

Hemolysin 

Complement 


Hemolysis  occurs  in  this  reaction  because  the  complement  is  not 
fixed  and  is  therefore  free  to  act  at  the  second  incubation. 

Each  of  the  factors  will  he  described  separately  in  the  order 
in  which  they  are  employed  in  the  test;  the  method  of  standard- 
ization will  be  explained ; and  finally  they  will  be  brought  to- 
gether in  proper  quantities  and  manner  to  illustrate  the  tech- 
nique followed  in  performing  the  test. 

The  Suspect'9  c Serum.  The  blood  of  the  animal  to  be  tested 
is  drawn  from  the  jugular  vein  by  means  of  a medium  sized  hy- 
podermic needle  or  a capillary  trochar.  Easily  excited  animals 
may  be  conveniently  controlled  with  a bull  lead.  Pressure 
with  the  thumb  over  the  jugular  will  cause  the  vein  to  engorge 
sufficiently  so  that  a stream  of  blood  will  flow  from  the  canula. 
If  many  blood  samples  are  required  at  one  time,  a small  rope 
drawn  tightly  about  the  neck  with  a knot  arranged  to  press  upon 
the  lower  portion  of  the  jugular,  will  help  in  this  operation. 
The  needles  are  kept  in  a large  mouthed  bottle  containing  a 1% 
solution  of  lvsol.  Lvsol  is  more  desirable  than  alcohol  in  that 
it  does  not  coagulate  blood  so  readily,  and  the  needles  are  there- 
fore easier  cleaned. 

The  blood  is  caught  in  sterile  test  tubes  holding  about  10  c.  c., 
or  in  large  mouthed  sterile  bottles  holding  about  1 ounce.  Each 
tube  or  bottle  is  labeled  and  properly  corked.  The  advantage 
of  test  tubes  is  that  they  may  be  placed  in  a centrifugal  machine 
later,  if  necessary,  to  separate  the  serum  from  the  blood  clot. 
Our  practice  has  been  to  loosen  the  clot  from  the  sides  of  the 


228 


Wisconsin  Research  Bulletin  No.  24 


tubes  and  place  them  in  the  ice  box  or  other  cool  place  over 
night  to  permit  the  serum  to  separate.  The  following  morning 
the  serum  is  ready  for  removal  to  smaller  test  tubes.  Finely 
drawn  pipettes  to  which  a small  rubber  nipple  is  fitted  will  be 
found  useful  for  this  purpose.  These  smaller  test  tubes  are 
convenient  to  handle  the  serum  in  while  it  is  undergoing  the 
process  of  inactivation. 

By  inactivation  is  meant  the  exposure  of  the  serum  to  a tem- 
perature from  55°  C.  to  56°  C.  for  one-half  hour  in  a water 
bath.  This  procedure  destroys  the  complement  of  the  cattle 
serum.  Complement,  as  will  be  more  fully  explained  later,  is  a 
thermo-labile  substance  present  in  all  normal  sera  but  in  vary- 
ing amounts.  Its  destruction  is  necessary  in  this  serum  so  that 
no  interference  may  be  experienced  in  the  test. 

If  it  is  necessary  to  hold  the  serum  for  some  days  before  test- 
ing, it  should  be  drawn  oft:  the  corpuscles,  and  preserved  by 
adding  to  each  9 c.  c.,  1 c.  c.  of  a solution  consisting  of  5 parts 
carbolic  acid,  10  parts  glycerin,  and  85  parts  of  a physiological 
sodium  chloride  solution  (0.9%).  Stored  in  an  ice  box  such 
carbolized  serum  should  retain  unchanged  for  several  weeks,  any 
immune  bodies  it  may  contain. 

The  Antigen.  In  the  complement  fixation  test  for  abortion, 
the  antigen  used  is  an  emulsion  of  the  abortion  bacilli  or  an  ex- 
tract of  the  same.  This  may  he  prepared  in  several  different 
ways.  (For  description  of  different  methods  see  page  245.)  The 
method  found  most  satisfactory  is  as  follows:  The  organisms 

are  grown  on  a serum-agar  slope  until  a heavy  growth  is  secured. 
This  culture  is  washed  off  with  5 to  15  c.c.  of  salt  solution  per  tube, 
depending  on  the  amount  of  growth.  It  is  then  preserved  by  ad- 
ding 10%  of  the  aforementioned  phenol-glycerin-salt  solution, 
and  shaken  in  a shaking  apparatus  for  24  hours.  The  best  re- 
sults have  been  obtained  with  a polyvalent  antigen,  i.  e.  one  made 
with  a number  of  strains  of  B.  abortus.  Such  prepared  antigens 
may  be  kept  for  several  months  if  stored  in  a cool,  dark  place. 
They  must,  however,  be  retitrated  from  time  to  time  to  establish 
their  strength.  Anti-complements  may  form  in  old  antigen ; 
fliese  may  be  readily  destroyed  by  heating  in  a water  bath  at 
56°  C.  for  20  minutes. 

The  Complement.  Fresh  blood  sera  from  practically  all  ani- 
mals contain  complement  in  varying  quantities.  On  this  ac- 


Complement  Fixation  Test  for  Contagious  Abortion  229 


count  care  must  be  exercised  to  guard  the  various  fluids  used  in 
the  work  against  possible  contamination  with  extraneous  serum. 
Guinea  pigs  are  best  adapted  to  furnish  complement  on  account 
of  the  more  constant  and  very  active  complemental  qualities  of 
their  serum.  Only  a small  quantity  is  required  in  conjunction 
with  'the  hemolysin  to  bring  about  hemolysis. 

The  blood  is  obtained  by  anesthetizing  the  animal  with  chloro- 
form. An  incision  is  then  made  transversely  across  one  side 
of  the  neck  with  a sharp  scalpel,  cutting  the  carotid  and  jugu- 
lar, but  not  the  trachea  or  oesophagus.  rflhe  blood  is  caught  in 
centrifuge  tubes.  A similar  incision  may  be  made  on  the  oppo- 
site side  of  the  neck  after  the  blood  ceases  to  flow  from  the  first 
incision.  As  soon  as  this  blood  clots  it  is  ready  to  be  centri- 
fuged for  the  recovery  of  the  serum. 

Complement  from  different  guinea  pigs  varies  somewhat  in 
activity.  It  is  also  very  sensitive  to  external  influences.  Wall 
states  that  complement  may  be  depended  on  to  keep  for  three 
days,  and  sometimes  longer.  In  our  opinion,  it  cannot  be  relied 
on  more  than  twenty-four  hours  after  it  has  been  drawn.  Com- 
plement twelve  to  fifteen  hours  old  has,  however,  been  found  to 
be  more  active  than  either  freshly  drawn  samples  or  samples 
older  than  this.  It  is,  therefore,  advisable  to  draw  the  blood  on 
the  evening  of  the  day  before  the  complement  is  required  for 
use  and  place  it  in  the  refrigerator  over  night.  The  titre  is  de- 
termined the  following  mo.rning  as  per  Table  I. 

The  Hemolysin.  The  power  which  the  blood  serum  of  an  ani- 
mal of  one  species  acquires  to  dissolve  the  red  blood  corpuscles 
of  an  animal  of  another  species  when  injected  with  such  cor- 
puscles, has  been  known  for  some  time.  In  the  process  of  disso- 
lution, the  hemoglobin  or  red  coloring  matter  of  the  corpuscles  is 
liberated.  The  process  is  known  as  hemolysis,  while  the  sub- 
stances which  effect  the  solution  of  the  corpuscles  are  called 
hemolysins. 

For  the  production  of  hemolysin  in  this  work,  a rabbit  is  im- 
munized against  horse  corpuscles.  The  procedure  to  be  followed 
in  procuring  and  washing  the  horse-blood  corpuscles  is  fully  des- 
cribed on  page  232. 

We  cannot  express  the  necessity  of  properly  washing  the  cor- 
puscles better  than  Bureau  of  Animal  Industry  Bulletin  136, 14 

14  Mohler,  J.  R.  and  Eichorn,  A.  The  Diagnosis  of  Glanders  by  Com- 
plement Fixation.  1911. 


230 


Wisconsin  Research  Bulletin  No.  24 


which  has  the  following  to  say  in  this  regard:  “The  washing 

of  the  blood  corpuscles  must  be  thoroughly  carried  out,  inas- 
much as  the  presence  of  even  traces  of  serum  adhering  to  the 
corpuscles  may  cause  difficulty  in  obtaining  satisfactory  results. 
If  rabbits  were  injected  with  red  blood  corpuscles  containing  a 
small  quantity  of  serum,  the  rabbits  would  develop,  not  only 
antibodies,  or  immune  bodies,  but  also  coagulins  and  anticom- 
plements, and  the  presence  of  these  substances  would  give  rise 
to  difficulties  in  demonstrating  the  presence  or  absence  of  a com- 
plete hemolysis.  Furthermore,  if  blood  corpuscles  containing 
even  traces  of  serum  were  used  in  the  tests,  it  might  produce  a 
fixation  of  the  complement,  and  thereby  give  rise  to  errors.  Such 
errors  would  occur  particularly  if  the  hemolytic  action  of  the 
rabbit  serum  was  not  very  high. 9 ? 

Equal  parts  of  the  washed  corpuscles  and  salt  solution,  heated 
to  the  body  temperature  (37.5°  C.),  are  mixed.  Fourteen  cubic 
centimeters  of  this  suspension  are  injected  intraperitoneally. 
At  the  end  of  seven  days  20  c.  c.  are  similarly  administered, 
and  after  another  seven  days  24  c.  c.  more. 

From  six  to  ten  days  after  the  last  injection  a sample  of  blood 
is  taken  from  an  ear  vein  of  the  rabbit  for  examination.  This 
blood  serum  should  possess  the  power  of  dissolving  the  red 
blood  corpuscles  of  the  horse.  In  other  words,  it  should  be 
hemolytic  for  horse  corpuscles. 

The  titre  of  this  rabbit  serum  or  hemolysin,  as  it  is  now 
termed,  is  next  established  according  to  Table  II.  If  it  is  found 
to  possess  sufficient  hemolytic  power  for  use,  the  rabbit'  is  sub- 
jected to  a further  bleeding.  One  ntethod  is  to  draw  the  blood 
from  the  ear  veins  into  test  tubes  which  will  fit  the  centrifuge. 
The  rabbit  is  not  killed  in  this  instance  and  may  be  used  later 
to  furnish  serum  by  additional  injections  with  horse  corpuscles. 
One  subsequent  injection  will  usually  stimulate  a further  produc- 
tion of  hemolytic  bodies.  In  case  the  blood  does  not  flow  freely 
from  the  severed  vein,  the  application  to  the  base  of  the  ear,  of 
a pledget’  of  absorbent  cotton  saturated  with  hot  water  will  be 
found  useful  to  bring  about  an  engorgement  of  the  vessels.  It 
appears  almost  needless  to  mention  the  necessity  of  shaving  and 
disinfecting  the  ear  prior  to  the  bleeding. 

The  other  method  requires  the  destruction  of  the  animal.  It 
is  chloroformed  and  the  thoracic  cavity  is  opened  under  aseptic 


Complement  Fixation  Test  for  Contagious  Abortion  231 

conditions.  The  heart  is  punctured  to  allow  the  blood  to  escape 
into  the  mediastinum,  from  which  it  is  at  once  drawn  by  sterile 
pipettes  and  placed  in  test  tubes  to  be  centrifuged. 

The  serum  is  now  recovered  and  preserved.  Here  again,  either 
of  two  methods  may  be  followed.  The  first  consists  of  drawing 
the  serum  into  small  sealed  tubes  with  a capacity  of  about  2 c.  c. 
each.  No  preservative  is  added,  so  strict  observance  of  the  rules 
governing  asepsis  is  necessary.  In  the  second  method  the  hemo- 
lytic serum  is  placed  in  10  c.  c.  test  tubes  and  mixed  with  10% 
of  the  phenol-glycerin-salt  solution.  Later  it  may  be  drawn 
into  the  smaller  tubes  and  sealed. 

Inactivation,  the  next  step,  is  carried # out  by  heating  in  a 
water  bath  at  56°  . C.  for  a half  hour.  The  hemolysin  is  stored 
in  the  icebox  where  it  will  keep  for  some  weeks.  The  titre 
should  be  re-established  every  two  weeks  or  thereabout,  as  the 
serum  in  some  cases  has  been  found  to  lose  its  activity  quite 
rapidly  after  storage. 

In  order  to  avoid  unpleasant  interference  in  hemolysin  pro- 
duction it  is  well  to  start  two  or  three  rabbits  at  the  same  time. 
Occasionally  a rabbit'  is  not  adapted  to  the  production  of  hemo- 
lytic substances;  in  other  cases  death  may  result  from  the  in- 
jections or  other  causes. 

At  one  time  it  was  thought  that  the  blood  serum  of  a certain 
species  of  animals,  after  they  had  been  injected  with  the  ery- 
throcytes of  another  species  would  hemolize  the  erythrocytes  of 
all  members  of  this  last  species  and  of  some,  but  not  all,  other 
species.  We  have  found  that  this  does  not  always  hold  true, 
at  least  in  so  far  as  horses  are  concerned.  Ehrlich,  Morgenroth 
and  others  in  their  experiments  with  goats  were  able  to  demon- 
strate a difference  in  similar  cells  of  the  same  species. 

We  have  shown  that  the  hemolysins  stimulated  in  a rabbit  by 
the  injection  of  the  red  blood  cells  of  a certain  horse  are  not 
specific  for  all  horses.  In  a series  of  tests  in  which  the  red 
blood  corpuscles  from  a number  of  different  horses  were  used 
with  a hemolysin  produced  by  injecting  a rabbit  with  red  cells 
from  a certain  horse,  approximately  50%  resulted  in  complete 
hemolysis.  In  the  balance  the  reaction  was  partially  or  wholly 
inhibited,  showing  that  not  all  horses  furnish  red  cells  of  uni- 
form character.  For  this  reason  the  same  horse  should  be  used 
throughout  this  work.  Otherwise,  unreliable  results  might  be 


232 


Wisconsin  Research  Bulletin  No.  24 


obtained  that  would  invalidate  all  tests  in  which  such  corpuscles 
were  employed. 

The  Washed  Red  Blood  Horse  Corpuscles.  The  red  blood  cor- 
puscles  of  a horse  are  employed  in  the  preparation  of  the  hemoly- 
sin, also  as  an  essential  factor  in  the  test  proper.  The  horse  has 
been  found  to  be  the  most  suitable  animal  from  which  to  obtain 
blood  because  no  restraint,  other  than  a twitch,  is  necessary  when 
small  quantities  are  drawn.  The  goat  or  sheep  may  be  em- 
ployed for  this  purpose  if  found  desirable. 


lhe  jugular  vein  is  tapped  in  a manner  similar  to  that  al- 
ready described  with  cattle.  The  place  for  the  insertion  of  the 
needle  should  be  disinfected  with  a pledget  of  absorbent  cot- 
ton moistened  with  alcohol.  A bottle  containing  small  glass 
beads  for  defibrination  of  the  blood  is  provided  to  catch  such 
quantity  as  required.  The  blood  is  defibrinated  by  shaking  the 
bottle  for  a few  minutes,  after  which  it  is  filtered  through  thin 
layers  of  absorbent  cotton  or  sterile  gauze  into  test  tnbes  of 
about  10  c.  c.  capacity.  These  are  placed  in  a centrifuge,  with 
a speed  of  2000-3000  revolutions  per  minute,  until  all  the  cor- 
puscles are  thrown  down.  The  supernatant  serum  is  pipetted 
off,  and  a corresponding  amount  of  sterile  physiological  salt  so- 
lution added  to  remove  the  remaining  traces  of  serum.  This 
washing  should  be  repeated  four  times,  in  order  to  dispose  of  all 
the  serum,  a very  necessary  procedure. 


Titration  of  the  Complement 

Tn  tlle  table  aml  illustrating  the  titration  of  the  com- 

plement, as  well  as  in  those  for  the  other  components,  actual  re- 
sults as  usually  obtained  in  practice  are  given,  in  order  to  give 
the  reader  a clear  conception  of  the  reaction.  The  various  quan- 
tities of  the  fluids  thus  determined  are  later  brought  together 
in  the  test  of  the  suspected  animal's  serum.  It  must  be  under- 
stood that  these  results  are  those  most  frequently  seen  in  prac- 
tice; however,  decided  variations  sometimes  occur. 

As  has  been  stated,  the  complement  is  derived  from  the  blood 
of  guinea  pigs.  We  aim  to  establish  the  smallest  amount  which, 
with  a definite  quantity  of  hemolysin,  will  induce  a complete 
hemolysis  of  0.5  c.  c.  of  a 1%  suspension  of  the  horse-blood  cells 
in  salt  solution.  Care  must  be  observed  in  all  these  titrations 
that  the  exact  amounts  of  the  different  fluids  are  used,  and  that 


) 


Wisconsin  Research  Bulletin  No.  24 


FIGURE  3.  TITRATION  OF  THE  COMPLEMENT 
Example'  of  results  usually  obtained. 


Complement  Fixation  Test  for  Contagious  Abortion  233 


the  dilutions  are  made  according  to  directions.  Carelessness 
will  lead  to  results  entirely  at  variance  with  actual  facts.  The 
pipettes  required  in  the  test  are  of  three  sizes,  viz.,  0.1  c.  c. 
graduated  to  0.01  and  0.001,  1.0  c.  c.  graduated  to  0.1  and  0.01, 
and  5.0  c.  c.  graduated  to  0.1.  All  glassware  should  be  care- 
fully cleansed.  Rinsing  all  tubes  and  pipettes  in  distilled  water 
just  before  use  is  recommended  by  the  most'  successful  techni- 
cians. Five  tenths  c.  c.  of  the  complement  mixed  with  2 c.  c. 
of  the  salt  solution  is  a sufficient'  amount  of  the  complement  for 
this  titration.  Two  tenths  c.  c.  of  the  diluted  hemolysin  is  as- 
sumed to  represent  the  quantity  of  this  substance  required. 
Seven  test  tubes  of  about  6 c.  c.  capacity  are  arranged  in  a rack 
and  into  each  are  carefully  measured  different  amounts  of  the 
necessary  components  as  per  Table  I. 


table  i.  titration  of  the  complement 


Tube 

1 

2 

3 

4 

5 

6 

r 

c.c. 

c.c. 

C.C. 

c.c. 

c.c. 

C.C. 

c.c. 

NaCl  solution  a 

1.5 

1.5 

t.5 

1.5 

1.5 

1.5 

1.5 

Hemolysin  b 

0.2 

0.2 

0.2 

0.2 

0.2 

0.2 

0.0 

Suspension  blood  corpuscles  c 

0.5 

0.5 

0.5 

0.5 

0.5 

0.5 

0.5 

Complement  d 

0.02 

0.04 

0.06 

0.08 

0.1 

0.0 

0.1 

Result  after  li  hours  e 

* 

* 

+ 

+ 

+ 

- 

- 

a 0.9%  sodium  chloride  solution. 
b 1%  dilution  hemolysin  in  sodium  chloride  solution. 

c,  ono/SUS£len?ion  washed  horse-blood  corpuscles  in  sodium  chloride  solution, 
a 40%  solution  complement  in  sodium  chloride  solution  (0.5  c.c.  complement  to 
2c.c.  salt  solution.) 

e -f  sign  indicates  complete  hemolysis;  — sign  indicates  no  hemolysis;  * signifies 
a variable  reaction  according  to  the  activity  of  the  complement. 

Shake  each  tube  and  place  the  rack  in  the  incubator  for  1 
hours,  then  read  the  results.  The  titre  of  the  complement  is 
represented  by  the  smallest  quantity  which  completely  dissolves 
the  red  corpuscles  and  leaves  a clear  red  solution.  Some  one 
of  the  first  five  tubes  will  contain  this  quantity.  In  the  titra- 
tion of  the  sample  from  which  Figure  3 was  taken,  tube  3 rep- 
resents the  titre.  Tubes  6 and  7 are  controls;  6 should  show 
no  hemolysis  as  no  complement  is  present;  7 is  a control  for  the 
guinea  pig  serum  to  ascertain  that  it  does  not  contain  hemolytic 
substances;  all  rabbit  serum  is,  of  course,  excluded  from  this 
tube.  Complement  with  a lower  titre  than  0.08,  represented 
by  tube  4,  should  be  discarded. 


234 


Wisconsin  Kesearch  Bulletin  No.  24 


In  the  application  of  the  complement  binding  test,  one  and 
one-half  times  the  litre  of  the  complement,  as  found  by  the  above 
titration,  is  used.  In  order  to  facilitate  measurement  in  the 
further  tests,  the  undiluted  complement  and  the  salt  solution  are 
mixed  in  such  a way  that  each  0.1  c.  c.  of  the  dilution  contains 
the  correct  quantity  of  complement. 

Titration  of  the  Hemolysin 

The  object  of  this  titration  is  to  establish  the  proper  quan- 
tity or  unit  necessary  for  use  in  the  complement  fixation  test. 
A unit  of  hemolysin  is  the  smallest  quantity  which  will  bring 
about  the  complete  solution  of  0.5  c.  c.  of  a 1%  suspension  of 
horse-blood  corpuscles  in  the  presence  of  the  proper  quantity  of 
complement.  The  precaution  of  heating  fresh  hemolysin  to  56° 
C.  must  be  observed  before  diluting  it,  in  order  to  destroy  any 
complement  which  it  may  contain  and  which  would  in  conjunc- 
tion with  the  hemolytic  bodies  be  capable  of  stimulating  hemoly- 
sis. A series  of  eight  test  tubes  is  arranged.  Into  each  is 
measured  1.5  c.  c.  of  salt  solution,  then  the  diluted  hemolysin, 
followed  by  the  suspension  of  horse-blood  corpuscles  and  the 
solution  of  the  complement  as  indicated  in  Table  II. 


table  ii.  titration  of  the  hemolysin 


Tube 

1 

2 

3 

4 

5 

6 

7 

8 

c.  c. 

c.  c. 

c.  c. 

c.  c. 

c.  c. 

c.  c. 

c.  c. 

c.  c. 

NaCl  solution  a 

1.5 

1.5 

1.5 

1.5 

1.5 

1.5 

1.5 

1.5 

Hemolysin  b 

0.02 

0.03 

0.05 

0.1 

0.15 

0.25 

0.15 

0.0 

Suspension  blood  corpuscles  c 

0.5 

0.5 

0.5 

0.5 

0.5 

0.5 

0.5 

0.5 

Complement  cL 

0.1 

0.1 

0.1 

0.1 

0.1 

0.1 

0.0 

0.1 

Result  after  11  hours  e 

* 

* 

+ 

+ 

+ 

+ 

— 

— 

a 0.9%  sodium  chloride  solution. 

l>  1%  dilution  hemolysin  in  sodium  chloride  solution. 

c 1%  suspension  washed  horse-blood  corpuscles  in  sodium  chloride  solution. 
d Complement  of  known  litre  diluted  so  that  0.1  c.  c.  contains  1*  times  the  estab- 
lished Quantity. 

e + sign  indicates  complete  hemolysis:  — sign  indicates  no  hemolysis;  * signifies  a 
variable  reaction  according  to  the  activity  of  the  hemolysin. 

Shake  each  tube  well,  place  the  rack  in  the  incubator  at  37°  C. 
for  I1/?  hours,  after  which  a reading  is  made  (See  Figure  4). 
The  standard  or  titre  of  the  hemolysin  is,  as  already  stated,  the 
smallest  quantity  which  completely  dissolves  the  red  blood  cor- 


FIGURE  4.  TITRATION  OF  THE  HEMOLYSIN 
Example  of  results  usually  obtained. 


Wisconsin  Research  Bulletin  No.  24 


Complement  Fixation  Test  for  Contagious  Abortion  235 


puscles  and  leaves  a clear  red  solution.  Hemolysis  may  appear 
in  any  of  the  first  six  tubes.  Tube  7 is  a control,  with  no  com- 
plement, to  demonstrate  that  the  hemolysin  is  properly  inac- 
tivated and  that  it  alone  without  the  addition  of  the  comple- 
ment does  not  have  a hemolytic  effect.  Tube  8 is  likewise  a 
control  to  prove  that  the  complement  without’  the  hemolysin 
will  not  provoke  hemolysis. 

The  hemolysin  is  used  in  triple  strength  for  all  further  titra- 
tions, so  as  to  insure  a sufficient  quantity  for  the  solution  of 
the  horse  corpuscles.  The  titre  of  the  diluted  hemolysin  should 
not  be  less  than  0.1  which  is  represented  in  tube  4.  Serum  with 
a lower  titre  than  this  should  not  be  used,  as  the  larger  quanti- 
ties that  would  be  required  might  interfere  with  the  reaction. 


Titration  of  the  Antigen 

Our  object  here  is  to  determine  the  smallest  quantity  of  an- 
tigen which  will  prevent  hemolysis  when  the  other  factors  are 
present  in  the  proper  quantities.  A series  of  twelve  small  test 
tubes  is  arranged  as  in  the  previously  described  titrations,  and 
the  different  fluids  are  added  as  indicated  in  Table  III.  It  will 

TABLE  III.  TITRATION  OF  THE  ANTIGEN 


Tube 

- 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

c.  c. 

c.  c. 

c.  c. 

c.  c. 

c.  c. 

c.  c. 

c.  c. 

c.  c. 

c.  c. 

c.  c. 

e.  c. 

c.  c. 

NaCl  solution  a 

1.5 

1.5 

1.5 

1.5 

1.5 

1.5 

1.5 

1.5 

1.5 

1.5 

1.5 

1.5 

Positive  cow  serum  b 

0.02 

0.02 

0.02 

0.02 

0.02 

0.02 

0.02 

0.0 

0.0 

0.0 

0.0 

0.0 

Negative  cow  serum  c.... 

0.0 

0.0 

0.0 

0.0 

0.0 

0.0 

0.0 

0.02 

0.0 

0.0 

0.0 

0.0 

Antigen  d 

0.02 

0.03 

0.05 

0.1 

0.15 

0.2 

0.25 

0.15 

0.15 

0.2 

0.25 

0.3 

Complement  e 

0.1 

0.1  ' 

0.1 

0.1 

0.1 

0.1 

0.1 

0.1 

0.1 

0.1 

0.1 

0.1 

Incubate  for  li  hours,  then  add  the  hemolytic  system  as  below. 


Hemolysin  f 

Suspension  blood  corpus- 
cles cr 

0.2 

0.5 

0.2 

0.5 

0.2 

0.5 

0.2 

0.5 

1 0.2 
0.5 

0.2 

0.5 

0.2 

0.5 

0.2 

0.5 

0.2 

0.5 

0.2 

0.5 

0.2 

0.5 

0.2 

0.5 

Result  after  2 hrs  h 

* 

* 

- 

- 

- 

- 

- 

+ 

+ 

+ 

+ 

* 

0.9%  sodium  chloride  solution. 

Inactivated  cow  serum  known  to  contain  the  specific  antibodies, 
inactivated  serum  from  a cow  known  to  be  free  from  abortion  infection 
Emulsion  of  a culture  of  B.  abortus  in  carbolized  salt  solution. 
C°l^hetl^1^an°itynOVVn  titre  diluted  so  that  °*  1 c-  c-  contains  li  times  the  estab- 

Hli?heFauantity  Wn  titre  diluted  so  that  °-  2 c-  c-  contains  three  times  the  estab- 

1%  suspension  washed  horse-blood  corpuscles  in  sodium  chloride  solution. 

-t-sigri  indicates  complete  hemolysis;  — sign  indicates  no  hemolysis;  * signifies 
a variable  reaction  according  to  the  activity  of  the  antigen. 


236 


Wisconsin  Research  Bulletin  No.  24 


be  observed  that  an  incubation  is  required  to  bring  about  the 
desired  reaction  before  the  hemolytic  system  is  added. 

After  a further  incubation  of  two  hours  the  results  are  read 
according  to  the  degree  of  hemolysis.  (See  Figure  5).  Some 
one  of  the  first  seven  tubes  will  indicate  the  titre,  i.  e.,  the  first- 
one  of  the  series  which  shows  no  hemolysis.  In  the  illustra- 
tion, tube  3 containing  0.05  e.  c.  represents  the  titre.  It  is  always 
required  that  the  antigen  be  strong  enough  to  bind  the  comple- 
ment in  tube  3 containing  0.05  c.  c.  together  with  0.02  c.  c. 
serum  from  a known  positive  reactor,  an  antigen  of  lower  titre 
should  not  be  used.  Tube  8 with  the  negative  serum  should 
always  show  complete  hemolysis.  Tubes  9,  10,  11,  and  12  are 
to  demonstrate  that  the  antigen  in  each,  without  the  presence  of 
a serum  containing  the  specific  antibodies  will  not  prevent  he- 
molysis. Tube  12  with  0.3  e.  c.  antigen  ordinarily  should  not 
fix  the  complement'. 

The  quantity  of  the  antigen  as  determined  by  this  titration 
is  not  taken  for  further  work ; instead,  four  times  this  quantity 
is  used,  as  will  be  noted  in  Table  IV. 

Method  of  Performing  the  Complement  Fixation  Test 

All  animals  infected  with  the  abortion  bacillus  develop  cer- 
tain specific  anti-bodies  or  immune  bodies  in  their  blood.  These 
bodies  will  vary  in  quantity  and  quality  depending  upon  the  in- 
dividual animal  as  well  as  the  character  of  the  infection.  For 
this  reason  it  is  necessary  to  use  different  quantities  of  the  sus- 
pect’s serum  in  the  final  test.  As  exact  amounts  are  required, 
special  care  is  necessary  in  measuring  them. 

It  is  understood  that  the  previously  described  titrations  have 
been  carried  out  and  the  titre  or  standard  of  each  component 
established.  All  the  substances  should  have  a high  titre.  A 
rack  with  eight  small  test'  tubes  is  arranged  for  each  animal  to 
be  tested.  Measure  the  required  amounts  of  salt  solution, 
serum,  antigen,  and  complement  into  the  respective  tubes,  shake 
each  tube  well,  and  incubate  for  one  and  one-quarter  hours. 
Then  add  the  hemolytic  system  according  to  Table  IV. 

After  the  test  tubes  have  been  incubated  again  for  two  hours 
the  results  should  be  read.  (See  Figures  6 and  7).  The  de- 
gree of  hemolysis  is  the  indicator.  The  first  four  tubes  are 
controls.  Tube  1 containing  salt'  solution  and  corpuscles  only, 


FIGURE  5.  TITRATION  OF  THE  ANTIGEN 
Example  of  results  usually  obtained. 


Wisconsin  Research  Bulletin  No.  24 


Complement  Fixation  Test  for  Contagious  Abortion  237 


should  show  no  hemolysis.  Hemolysis  should  always  occur  in 
tube  2,  as  the  complement  and  hemolysin  are  present  in  the 
proper  quantities  to  bring  about  the  solution  of  the  horse  cor- 
puscles. Tube  3 should  likewise  show  complete  hemolysis  be- 
cause there  is  no  antibody  present  to  bind  the  complement ; fur- 
ther, this  tube  demonstrates  that  the  antigen  in  the  quantity 
used  throughout  the  test  will  not  of  itself  bind  the  complement. 
Tube  4 is  to  show  that  the  antigen  will  not  stimulate  hemolysis 


TABLE  IV.  TEST  OF  THE  SUSPECTS  SERUM 


Tube 

1 

2 

3 

4 

5 

6 

7 

8 

c.  c. 

c.  c. 

c.  c. 

c.  c. 

■ 

c.  c. 

c.  c. 

c.  c. 

c.  c. 

NaCl  solution  a 

1.5 

1.5 

1.5 

1.5' 

1.5 

1.5 

1.5 

1.5 

Suspect’s  serum  b 

0.0 

0.0 

0.0 

0.0 

0.02 

0.01 

0.02 

0.04 

Antigen  c 

0.0 

0.0 

0.2 

0.2 

0.2 

0.2 

0.2 

0.2 

Complement  d 

0.0 

o.i 

0.1 

0.0 

0.1 

0.1 

0.1 

0.1 

Incubate  for  11  hours,  then  add  the  hemolytic  system  as  below. 


Hemolysin  e 

Suspension  blood  corpuscles  / 

0.0 

0.5 

0.2 

0.5 

0.2 

0.5 

0.2 

0.5 

0.2 

0.5 

0.2 

0.5 

0.2 

0.5 

0.2 

0.5 

Result  after  2 hours  g 

— 

+ 

+ 

* 

* 

* 

* 

a 0.9%  sodium  chloride  solution. 

h Undiluted  cattle  serum  previously  inactivated  for  30  minutes  at  56°  C. 

c Four  times  the  amount  established  at  the  antigen  titration. 

d Complement  of  known  titre  diluted  so  that  0.  1 c.  c.  contains  li  times  the  estab- 
lished Quantity. 

e Hemolysin  of  known  titre  diluted  so  that  0.2  c.  c.  contains  three  times  the  estab- 
lished Quantity. 

/ 1%  suspension  washed  horse  blood  corpuscles  in  sodium  chloride  solution. 

g p sign  indicates  complete  hemolysis;  _ sign  indicates  no  hemolysis;  * signifies  a 
variable  reaction  according  to  the  nature  of  the  serum. 

in  the  absence  of  the  complement.  Tubes  5 and  7 contain  the 
unknown  serum  and  are  alike.  They  should  show  hemolysis  if 
the  animal  is  not  infected  with  the  abortion  bacilli,  but  if  im- 
mune bodies  are  present  in  the  serum  these  tubes  will  both  re- 
main unchanged,  indicating  a complement  binding.  Tube  6 
with  a smaller  amount  of  the  suspect’s  serum  may  show  binding 
if  the  immune  bodies  are  present  in  sufficient  quantities ; a very 
active  serum  will  do  this.  Tube  8 contains  all  the  components 
with  an  excess  of  the  suspect’s  serum;  no  hemolysis  will  take 
place  if  the  animal  is  diseased,  hemolysis  if  no  immune  bodies  are 
present. 


238  Wisconsin  Research  Bulletin  No.  24 

It  must  be  noted  that  occasionally  the  results  are  not  abso- 
lute, that'  is,  there  may  be  a partial  or  incomplete  hemolysis  in 
tubes  5 and  7.  This  is  taken  as  an  indication  that  the  anti- 
bodies are  not  present  in  sufficient  quantity  to  bind  the  comple- 
ment. In  such  a case  it  is  generally  assumed  that  the  animal 
in  question  has  become  infected  recently,  or  that  she  is  just  re- 
covering from  an  attack  of  the  disease.  In  either  instance  a 
retest  must  be  made  in  four  to  six  weeks  to  determine  positively 
which  condition  really  exists.  Tube  8 with  the  double  quantity 
of  the  suspect’s  serum  may  be  taken  as  a fairly  reliable  guide  in 
these  doubtful  reactions. 

A negative  reaction  does  not  necessarily  exclude  the  presence 
of  the  abortion  bacilli  in  the  animal’s  body.  The  infection  may 
have  occurred  so  recently  that  the  immune  bodies  have  not  been 
formed  in  sufficient  quantities  to  bring  about  the  reaction. 

In  some  cases  a positive  reaction  is  obtained  from  the  serum 
of  a pregnant  cow  or  heifer.  This  does  not  always  mean  that 
the  animal  will  abort,  for  it  lias  been  shown  that  abortion  is 
simply  incidental  to  the  infection.  Experimental  animals  Nos. 
1 and  6 (pages  240  and  241)  are  two  cases  which  illustrate  this 
point  very  nicely. 

Occasionally  anticomplements  will  form  in  an  otherwise  nat- 
urally negative  serum  after  standing  some  time.  It  is  therefore 
always  best  to  heat  old  serum  over  the  water  bath  for  20  min- 
utes at  56°  C.  to  destroy  the  anticomplemental  substances;  other- 
wise, in  a test  of  such  serum,  what  would  appear  like  a positive 
reaction  would  in  reality  be  negative. 

Unsatisfactory  results  usually  indicate  either  improperly 
prepared  materials,  or  defective  technique,  and  call  for  a repe- 
tition of  the  test.  To  facilitate  the  work  when  a large  number 
of  tests  are  to  be  made  the  same  day,  we  advise  mixing  the 
hemolysin  and  the  corpuscle  suspension  just  before  they  are  re- 
quired for  use  in  the  hemolytic  system. 

The  interpretation  of  the  reaction  may  be  summarized  as  fol- 
lows: ! 1 < 3 ! 

1.  Cattle  in  which  the  serum  shows  a complete  fixation  of  the 
complement  in  quantities  of  0.01  c.  c.  and  0.  02  c.  c.  are  or  have 
been  infected  with  abortion  bacilli. 

2.  Cattle  in  which  the  serum  gives  a complete  complement 
fixation  in  the  quantity  of  0.02  c.  c.  and  an  incomplete  fixation 


FIGURE  7.  THE]  COMPLEMENT  FIXATION  TEST 
Example  of  results  obtained  in  a negative  reaction. 


Wisconsin  Research  Bulletin  No.  24 


FIGURE  7.  THE  COMPLEMENT  FIXATION  TEST 
Example  of  results  obtained  in  a negative  reaction. 


Wisconsin  Research  Bulletin  No.  24 


Example  of  results  obtained  in  a positive  reaction. 


Wisconsin  Research  Bulletin  No.  24 


Complement  Fixation  Test  for  Contagious  Abortion  239 


in  the  0.01  c.  c.  amount,  also  are  or  have  been  infected  with  the 
abortion  bacilli. 

3.  Cattle  in  which  0.01  c.  c.  of  the  serum  shows  no  binding 
while  the  larger  quantity  gives  an  incomplete  binding  should  he 
considered  questionable  reactors  and  retested  after  four  to  six 
weeks. 

4.  Cattle  in  which  the  serum  shows  no  power  of  fixing  the 
complement  in  either  amount  should  be  considered  free  from  the 
infection. 

If  contagious  abortion  is  found  in  animals  of  a certain  herd, 
all  should  be  subjected  to  a retest  within  a reasonable  time.  Re- 
acting cattle  should  be  quarantined  or  otherwise  disposed  of, 
and  the  premises  properly  disinfected. 

The  accuracy  of  this  method  of  diagnosis  is  as  great  as  any 
method  based  upon  a biological  reaction.  Further  experimen- 
tation may  perhaps  suggest  some  modification  of  the  present 
technique.  Where  large  numbers  of  animals  are  to  be  tested, 
such  changes  would  be  acceptable  if  not  made  at  the  expense 
of  accuracy.  However,  at  present  we  find  it  quite  possible  to 
run  tests  on  50  or  more  animals  a day  without  undue  exertion. 

The  test  affords  a reliable  means  by  wThich  infected  animals 
may  be  detected.  Proper  methods  of  isolation  and  control  may 
then  be  instituted  by  which  the  disease  can  be  prevented  from 
spreading  to  noninfected  cows  and  heifers.  It  is  a qualitative 
and  not  a quantitative  test  and  simply  indicates  the  presence  or 
absence  of  the  specific  immune  bodies;  but  clinical  history  has 
so  closely  corroborated  the  test  that  we  may  assume  it  to  be  a 
reliable  guide  as  to  the  presence  or  absence  of  the  abortion  ba- 
cilli. 


ADDITIONAL  EXPERIMENTAL  WORK 
Purpose  of  Our  Studies 

The  experimental  work  undertaken  by  us  was  first  directed 
toward  establishing  the  reliability  of  the  complement  fixation 
test.  After  this  wras  accomplished,  and  the  technique  of  manipu- 
lation perfected,  we  made  use  of  the  delicate  reaction  to  check 
up  other  experiments. 

Endeavor  has  been  made  to  find  some  practical  means  by 
which  contagious  abortion  might  be  controlled.  Among  other 


240 


Wisconsin  Research  Bulletin  No.  24 


things  it  is  of  course  of  utmost  importance  to  know  if  this  new 
test  was  rigidly  accurate  when  applied  to  the  same  animal  at 
stated  intervals;  also  whether  environmental  conditions  influ- 
enced such  reactions.  To  establish  certain  of  the  above  points 
the  following  experiments  have  been  carried  out. 

Consecutive  Tests  in  the  Infected  Herd 

The  herd  selected  as  most  suited  for  systematic,  testing  be- 
longed to  the  Experiment  Station  and  afforded  valuable  ex- 
perimental material,  due  to  the  fact  that  it  was  composed  of 
heifers  which,  had  never  calved  and  cows  which  were  known  to 
have  aborted.  There  were  ten  animals  in  the  “ infected”  herd 
which  was  under  quarantine  at  a separate  farm.  The  heifers 
had  been  unavoidably  exposed  to  the  infection  through  con- 
tact with  the  diseased  cows  in  January  1911.  A short  history 
of  each  animal  follows: 

History  of  Animals  in  the  Infected  Herd 

Heifer  1 (Cora)  dropped  a calf  at  full  term,  April  29,  1911. 
The  calf  was  apparently  normal  in  every  respect  but  died  in 
half  an  hour.  The  placenta  and  fluids  were  caught  in  sterile 
containers  and  immediately  taken  to  the  laboratory  for  exam- 
ination. Media  were  inoculated  but  no  growths  resembling  the 
abortion  bacilli  could  be  found.  The  heifer’s  blood  was  tested 
by  the  complement  fixation  method  in  July  and  gave  a positive 
reaction.  Eor  subsequent'  tests  see  Table  V.  This  heifer  has 
come  in  heat  at  irregular  periods  for  more  than  one  year  and 
has  been  served,  but  has  apparently  failed  to  conceive,  indicat- 
ing a pathological  condition  of  the  genital  organs  due  to  the 
infection.  Possibly  conception  took  place  at  the  first  or  second 
service  and  a second  abortion  occurred  so  early  in  gestation 
that  it  escaped  the  attention  of  the  attendant. 

Heifer  2,  registry  No.  31002,  commenced  to  give  considerable 
milk  July  1,  1911,  although  she  had  never  calved,  at  which  time 
she  was  given  the  test  and  responded  positively.  On  August  1, 
1911,  she  aborted  thus  confirming  our  diagnosis.  Her  fetus 
was  recovered  and  taken  to  the  laboratory  where  cultures  were 
made  from  the  stomach  Contents.  Doctor  Larson15  found  abor- 

15  Dr.  W.  P.  Larson,  now  with  the  University  of  Minnesota,  was  asso- 
ciated with  us  in  this  work  for  a few  weeks  in  the  summer  of  1911.  We' 
take  this  opportunity  to  acknowledge  our  indebtedness  to  him. 


Complement  Fixation  Test  for  Contagious  Abortion  241 

tion  bacilli  in  large  numbers  on  the  serum-agar  culture  medium 
and  prepared  an  antigen  from  the  organisms.  This  organism 
he  later  demonstrated  to  be  identical  with  the  Bang  bacillus. 

Heifer  3 (Agnes),  a pure  bred  Holsfein,  aborted  February 
19,  1911.  First  test  for  abortion  was  made  in  July,  1911,  when 
the  reaction  was  positive.  Further  tests  showed  the  reactions 
noted  in  Table  V. 

Heifer  4,  an  Ayrshire,  aborted  May  29,  1911.  A peculiar 
feature  about  this  heifer  was  the  persistent  negative  reaction 
which  she  gave  for  some  months  after  abortion,  and  the  event- 
ual appearance  of  the  immune  bodies  as  determined  by  the  test. 
We  are  led  to  believe  that  abortion  in  this  case  was  due  to  acci- 
dental causes. 

Heifer  5,  a pure  bred  Jersey  calf,  dropped  March  10,  1911, 
about  six  weeks  prematurely.  The  dam  was  later  found  to  be 
a positive  reactor.  As  a calf  this  animal  sucked  the  infected 
mother  and  was  kept  in  the  infected  herd.  In  spite  of  these 
excellent  possibilities  for  acquiring  the  infection-  the  tests  indi- 
cate that  no  antibodies  could  be  demonstrated  in  her  serum 
until  she  was  about  one  year  old. 

Heifer  6,  registry  No.  31004,  was  dropped  November  5,  1909, 
and  served  May  20,  1911.  Like  heifer  7 she  ran  with  infected 
animals  from  January  to  October,  1911.  No  signs  of  abortion 
were  ever  noted.  She  was  selected  as  a good  animal  to  place 
under  the  carbolic  acid  treatment,  and  so  was  isolated  with 
heifers  5 and  7 in  October.  The  medication  consisted  of  sub- 
cutaneous injections  of  8 c.  c.  of  a 3%  solution  of  the  acid  every 
ten  days.  On  January  30,  1912,  the  strength  of  the  carbolic 
acid  solution  was  increased  to  5%.  She  calved  February  19, 
1912.  Portions  of  the  placenta  were  immediately  taken  to  the 
laboratory,  also  some  fluid  from  the  parturient  uterus;  media 
were  inoculated  and  the  organism  isolated  from  scrapings  of  the 
cotyledons.  An  antigen  prepared  from  this  growth  gave  a typi- 
cal binding  when  titrated  against  the  serum  of  a known  positive 
reactor.  The  results  of  experimental  injections  of  the  recovered 
organism  into  the  veins  of  a pregnant  ewe  are  noted  on  page  244. 
There  can,  therefore,  be  no  question  that  the  abortion  bacilli  are 
present  in  this  heifer.  It  is  interesting  to  note  that  a test  of  the 
blood  serum  obtained  from  the  calf  when  it  was  25  days  old 
gave  a complete  fixation  of  the  complement.  This  case  demon 


242  Wisconsin  Research  Bulletin  No.  24 

to****.-  - 

strates  very  nicely  that  cows  may  harbor  - the  infectious  agent, 
yet  not  always  abort.  Such  animals  are,  however,  dangerous 
sources  of  infection  and  may  transmit  the  disease  to  others. 

Heifer  7,  registry  No.  31001,  was  dropped  October  27,  1909, 
and  served  about  April  17,  1911.  She  ran  with  infected  ani- 
mals from  January  to  October,  1911,  when  she  was  isolated  with 
heifers  5 and  6 at  which  time  carbolic  acid  treatment  was  in- 
augurated. The  dose  in  this  case  was  2 c.  c.  of  the  pure  acid 
diluted  in  water  and  placed  on  the  bran  mash  every  other  day, 
night  and  morning.  On  January  30,  1912,  the  dosage  of  car- 
bolic acid  was  increased  to  3 c.  c.  Abortion  occurred  December 
21,  1911.  The  fetus  and  fetal  membranes  were- taken  to  the 

TABLE  V.  SUMMARY  OP  CONSECUTIVE  TESTS  IN  THE  INFECTED  HERD 

+ sign  indicates  positive  reaction;  — sign  indicates  negative  reaction;  ? mark  de- 
notes atypical  reaction. 


No. 

1911 

J uly  test 

1911 

Oct.  test 

1911 

Dec.  test 

1912 

Jan.  test 

1912 

Mar.  test 

1912  1 

Apr.  test 

Abortion  history 

1.... 

+ 

+ 

+ 

+ 

Calved  4,  29/11 

2.... 

+ 

+ 

+ 

+ 

+ 

Aborted  8 1 11 

3.... 

+ 

+ 

+ 

+ 

+ 

+ 

Aborted  2 19  11 

4.... 

— 

— 

? 

+ 

+ 

+ 

Aborted  5,  29  11 

5.... 

— 

- — 

— 

— 

v 

— 

Dropped  3 10  11 

6. . . . 

• — 

? 

? 

+ 

+ 

+ 

Calved  2 19  12 

7.... 

— 

? 

+ 

+ 

+ < 

+ 

Aborted  12  21  11 

8.... 

+ 

+ 

+ 

+ 

+ 

+ 

Probably  aborted  8 11 

9.... 

+ 



+ 

4- 

Aborted  years  ago 

10.... 

— 

+ 

+ 

Bull 

laboratory  and  different  media  inoculated  from  the  stomach 
contents,  heart’s  blood,  and  with  scrapings  from  the  placen- 
tal cotyledons.  Cultures  of  the  abortion  bacillus  were  obtained 
on  the  serum-agar  plates  from  the  stomach  contents  of  the  fetus. 
T1  lese  were  eventually  grown  in  pure  culture  and  used  for  the 
preparation  of  antigen  which  proved  to  be  specific  when 
titrated  against  serum  from  known  positive  reactors,  thus  es- 
tablishing the  identity  of  the  organism. 

Heifer  8,  registry  No.  31003,  was  dropped  November  4,  1909, 
and  bred  in  May,  1911.  While  at  pasture  in  August,  1911,  she 
was  seen  to  have  a slight  vaginal  discharge  which  was  attributed 
to  oestrum  so  no  attention  was  given  the  animal.  Recurrence 
of  oestrum  was  noted  in  October  and  again  in  December.  A 
positive  reaction  was  given  with  her  serum  in  July  so  it  seems 
likely  that  this  heifer  aborted  at  pasture.  This  belief  is  sub- 


Complement  Fixation  Test  for  Contagious  Abortion  243 

stantiated  by  recurrence  of  oestrum  and  the  fact  that  no  signs 
of  pregnancy  have  since  been  visible. 

Cow  9,  an  aged  grade  Hereford,  aborted  some  years  ago  (ex- 
act date  not  known).  This  cow  had  retained  placenta,  and 
milk  fever  November  17,  1910,  but  calved  normally  next'  season. 

Bull  10,  a pure  bred  Guernsey,  used  exclusively  for  service  in 
the  infected  herd. 

The  dates  of  and  reactions  obtained  from  the  consecutive  tests 
on  these  animals  are  given  in  Table  V. 

Discussion  of  Results  with  Consecutive  Tests 

Among  the  interesting  points  brought  out  by  this  experiment 
are  the  following : 

(1)  The  persistence  of  the  immune  bodies  longer  than  a year 
after  abortion  occurred,  e.  g.,  No.  3. 

(2)  The  gradual  appearance  of  these  bodies  in  different  cases, 
e.  g.,  Nos.  4,  6,  7,  10. 

(3)  The  fact  that  No.  5,  a calf,  did  not  show  the  immune 
bodies  up  to  a year  old,  although  she  was  dropped  prematurely, 
and  was  in  constant  association  with  infected  animals. 

(4)  The  accuracy  with  which  the  test  detected  infected  ani- 
mals, (a)  even  before  any  clinical  signs  of  abortion  were  noted, 
e.  g.,  Nos.  6 and  7 ; (b)  in  the  case  of  one,  No.  8,  which  was  sup- 
posed to  be  safely  pregnant,  but  which  had  probably  aborted. 

(5)  The  reaction  given  by  the  herd  bull,  No.  10,  after  associa- 
tion with  infected  cows. 

Laboratory  Experiments 

Twenty  placental  membranes  and  fetuses  have  been  examined 
bacteriologically.  The  abortion  organism  was  isolated  from  thir- 
teen of  the  specimens.  Some  of  the  others  were  accidental  abor- 
tions, as  was  conclusively  demonstrated  by  subsequently  testing 
the  cows’  blood  serum.  Microscopical  identification  alone  has 
not  been  found  reliable. 

In  order  to  further  demonstrate  that  the  organisms  recovered 
were  the  cause  of  abortion  a number  of  rabbits  were  injected 
intraperitoneally  and  intravenously  with  emulsions  of  different 
strains.  These  does  aborted  within  a few  hours  after  the  in- 
jections which  accounts  for  the  negative  results  obtained1  from 


244 


Wisconsin  Research  Bulletin  No.  24 


the  cultural  examinations  of  the  stomach  contents  and  heart’s 
blood  of  the  fetuses,  and  the  uterine  contents  of  the  does. 

An  especially,  interesting  experiment  was  the  one  carried  out 
on  a pregnant  ewe  as  follows : 

Ewe  769  was  bred  November  24,  1911.  On  March  17,  1912, 
thirty-five  days  before  she  was  due  to  lamb,  she  received  by  in- 
trajugular injection  4 c.  c.  of  an  emulsion  of  abortion  bacilli  re- 
covered from  heifer  6.  This  emulsion  was  made  by  washing  the 
growth  from  a 10-day  serum-agar  slope  with  20  c.  c.  of  normal 
salt  solution,  and  was  rich  in  abortion  bacilli.  On  April  2,  sev- 
enteen days  after  the  injection,  the  ewe  aborted  twin  lambs.  One 
never  breathed,  as  indicated  by  lung  tissue  which  was  liver-like 
in  appearance  and  sank  in  water,  showing  that  the  alveoli  had 
never  been  filled  with  air.  The  other  lived  for  a few  hours  but 
was  very  weak  and  undersized.  Plates  were  inoculated  from  the 
umbilicus  and  stomach  contents  of  both  fetuses,  also  from  a cot- 
yledon of  the  placenta.  Stained  material  from  the  stomach  con- 
tents revealed  a small  organism  which  appeared  like  the  abortion 
bacillus.  Typical  colonies  were  obtained  on  the  plates  which  had 
been  seeded  from  the  stomach  and  umbilicus,  thus  satisfying 
Koch’s  postulates.  The  ewe’s  blood  serum  was  tested  one  week 
after  abortion  occurred  and  gave  a decided  complement  fixation, 
indicating  the  presence  of  a large  quantity  of  the  specific  im- 
mune bodies.  1 

I . L.  — . L 

Resistance  of  Abortion  Bacilli 

B.  abortus  seems  to  have  considerable  resistance  and  will  live 
for  some  time  outside  the  animal  body.  Bang  and  Stribolt  were 
able  to  isolate  the  organism  in  pure  culture  from  two  fetuses 
which  had  been  dead  five  months  and  nine  months,  respectively. 
The  uterine  exudate  kept  on  ice  contained  living  organisms  after 
seven  months. 

Freezing  does  not  kill  this  organism.  On  February  13,  1912, 
an  aborted  fetus  was  forwarded  from  Columbus,  Wis.  Upon 
arrival  at  the  laboratory  the  calf  was  frozen  solidly;  however,  the 
abortion  bacilli  were  isolated  by  the  plate  method.  On  Feb- 
ruary 29  the  fetal  membranes  of  an  aborted  fetus  were  forwarded 
from  Beloit,  Wis.  The  membranes  were  frozen  as  hard  as  a cake 
of  ice;  nevertheless,  upon  cultural  examination  the  abortion 
bacilli  were  found  alive  and  vigorous.  The  organisms  were  recov- 


Complement  Fixation  Test  for  Contagious  Abortion  245 

ered  in  pure  culture.  Later,  antigens  were  prepared  with  both 
strains  and  gave  a firm  binding,  when  titrated  against  a positive 
serum. 

Other  Methods  for  the  Preparation  of  Antigen 

Besides  the  method  previously  described  for  the  preparation 
of  the  abortion  antigen,  the  following  may  be  mentioned : 

A.  To  200  c.  c.  sterile  bouillon  in  a specially  blown  flask  are 
added  50  c.  c.  sterile  raw  blood  serum.  After  two  or  three  days 
incubation  to  determine  sterility,  it  is  inoculated  with  abortion 
bacilli.  Oxygen  is  allowed  to  bubble  through  the  liquid  to  drive 
off  at  least  90%  of  the  air ; then  it  is  incubated  10  to  12  days, 
after  which  it  is  carbolized  and  titrated  as  previously  described. 

B.  Serum  bouillon  containing  the  growth  of  abortion  bacilli, 
prepared  as  above,  is  centrifuged.  The  supernatant  liquid  is 
then  pipetted  off,  leaving  tbe  organisms  clumped  together  m the 
bottom  of  the  tube.  Physiological  salt  solution  is  added  to  two- 
thirds  of  the  original  volume.  This  suspension  of  abortion 
bacilli  in  salt  solution  is  very  good  antigen,  more  satisfactory  in 
some  respects  than  that  referred  to  above  because  the  foreign 
horse  serum  has  been  removed. 

C.  Another  method  which  has  been  fairly  satisfactory  is  to 
centrifuge  in  the  manner  described  under  B.  The  salt  solution 
is  added,  the  suspension  again  thrown  down,  and  the  supernatant 
solution  pipetted  off.  This  is  repeated  three  times  until  all  of 
the  serum  bouillon  is  removed.  The  mass  is  then  ground  with 
sterile  sand  or  pulverized  glass  until  the  organisms  are  broken 
up.  Salt  solution  is  added  to  one-half  the  original  volume  and 
the  mixture  placed  on  ice  for  12  to  24  hours.  The  cellular  ele- 
ments, sand,  etc.  are  removed  by  means  of  a porcelain  filter. 
Antigen  prepared  in  this  manner  has  few  advantages  over  that 
described  above.  It  is  a clear  solution,  and  a little  nicer  to 
work  with  on  that  account,  and,  if  properly  employed,  will  give 
satisfactory  results. 

It  was  observed  that  some  antigens,  prepared  by  the  use  of 
serum  bouillon,  would  bind  the  complement  in  the  absence  of 
the  positive  serum,  in  such  small  quantities  as  to  make  their  use 
in  the  test  prohibitive.  It  was  thought'  that  the  raw  horse  serum 
in  the  mixture  might  be  responsible  for  this.  Accordingly  anti- 
gens which  bound  the  complement  were  treated  as  described  urn 


246 


Wisconsin  Research  Bulletin  No.  24 


der  C,  but  this,  while  eliminating  the  raw  serum,  did  not  do  away 
with  the  binding.  When  sterile  serum  and  bouillon  are  mixed 
and  incubated  there  ofttimes  a copious  precipitate.  A quan- 
tity of  this  precipitate  was  withdrawn  from  a flask  before  inocu- 
lating with  the  abortion  bacilli.  The  content  of  the  flask  was 
sterile  as  shown  by  cultural  and  microscopical  examination. 
This  precipitate  bound  the  complement  in  .04  to  .06  c.  c.  and  par. 
tially  in  0.02  c.  c.,  showing  that  it  was  responsible  for  the  un- 
pleasant fixation  noted  above. 

b ixation  of  the  complement  has  not  been  observed  in  antigens 
prepared  as  described  on  page  228,  in  any  amount  up  to  0.3  c. 
c. ; consequently  that  method  is  to  be  recommended  as  the  most 
satisfactory. 

Field  Experiments 

In  order  to  show  the  practical  value  of  this  new  diagnostic 
method,  it  was  necessary  to  conduct  tests  in  different  herds. 
Concrete  examples  of  the  conditions  under  which  the  tests  were 
made,  follow: 

A noted  pure  bred  dairy  herd  was  visited  with  the  object  of 
prescribing  preventive  and  curative  measures  for  white  scours 
in  the  calves.  The  disease  was  so  virulent  that  it  had  been  im- 
possible to  raise  calves,  twelve  having  died  within  a period  of 
two  months.  We  had  no  suspicion  of  contagious  abortion  at  this 
time,  as  the  calves  had  all  been  carried  full  term,  but  thought  it 
a good  opportunity  to  try  out  the  fixation  test'.  Consequently 
samples  of  blood  were  taken  from  each  of  the  six  cows  which  had 
recently  calved.  Upon  examining  the  blood  serum  it  was  found 
that,  with  one  exception,  all  responded  negatively  and  were  there- 
fore pronounced  free  from  contagious  abortion  infection.  The 
exception  was  a four  years  old  cow  which  gave  a most'  beautiful 
positive  reaction.  The  results  were  reported  to  the  owner  with 
the  request  that  he  furnish  us  information  as  to  her  history.  lie 
replied  that  the  cow  did  abort  in  1910,  as  did  a number  of  others 
associated  with  her  at  the  time. 

Another  instance,  which  illustrates  the  reliability7'  of  the  test 
even  more  conclusively,  was  a test  made  on  some  samples  for- 
warded by  Dr.  L.  A.  Wright  of  Columbus,  Wis.  His  letter  un- 
der date  of  Feb.  10,  1912,  states  the  facts  as  follows: 

Your  favor  just  at  hand  with  record  of  test  enclosed.  I did  not  give 
you  any  assistance  in  the  cases,  as  you  know  we  sometimes  see  things 


Complement  Fixation  Test  for  Contagious  Abortion  247 


wrongly  when  influenced  to  do  so  by  suggestions.  You  will  be  inter- 
ested and  pleased  to  know  that  No.  3,  positive,  has  since  aborted. 
No.  17  was  the  one  which  had  aborted.  I removed  the  afterbirth  at 
once  and  washed  her  out  and  she  has  had  carbolic  acid  every  two  days, 
in  teaspoonful  doses,  ever  since,  so  she  is,  and  was  at  the  time  we  sent 
blood  samples,  quite  clean.  No.  14  calved  since  we  sent  tubes,  appar- 
ently all  right,  but  she  is  an  old  cow  a*nd  was  well  advanced  in  preg- 
nancy before  she  was  brought  into  this  herd  and  may  have  harbored 
the  germs  without  causing  abortion.  Came  near  forgetting  to  tell  you 
that  I sent  you,  by  express,  yesterday,  the  aborted  calf  of  No.  13.  I 
froze  it  and  packed  it  i'ii  snow. 

The  calf  referred  to  in  the  above  letter  was  the  one  from  which 
cultures  were  obtained  as  noted  on  page  244. 

Mr.  II.  N.  Langley  of  Dousman,  Wis.,  writes  in  his  letter  of 
April  23,  as  follows: 

Your  test  for  abortion  corresponds  exactly  with  the  history  of  the 
cases.  We  had  little  reason  to  suspect  the  disease  in  numbers  1 and  2. 
Numbers  3 and  4 are  two  pure  breds  purchased  about  a year  ago: 
number  3 aborting  and  number  4 calving  prematurely  a few  months 
after  purchasing  them,  and  both  have  failed  to  get  with  calf  si'nce. 

These  are  but  three  of  many  instances  which  substantiate  the 
test  and  which  have  led  us  to  place  much  confidence  in  its  ac- 
curacy. 


TABLE  VI.  SUMMARY  OF  RESULTS  WITH  THE  COMPLEMENT  FIXATION 
Twenty-two  infected  herds  tested,  a 


Number 

of 

animals 

Posi- 

tive 

Nega- 

tive 

Reac 

Ques- 

tion- 

able 

tion 

Per 
; cent 
posi- 
tive 

Per 

cent 

nega- 

tive 

Per 

cent 

ques- 

tion- 

ab.e 

No  history  of  abortion 

301 

53 

238 

13 

17.4 

78.3 

4.3 

Known  aborters 

97 

81 

14 

2 

83.5 

14.4 

2.1 

Herd  bulls 

10 

1 

9 

0 

10.0 

90.0 

0.0 

Totals 

411 

135 

261 

15 

32.9 

63.5 

3.6 

a A very  few  of  these  animals  came  from  herds  where  no  infection  existed,  but  are 
included  for  the  purpose  of  this  work. 


Table  VI  shows  the  results  obtained  from  the  tests  on  cattle 
from  various  widely  separated  parts  of  the  state.  A brief  analy- 
sis reveals  the  fact  that  17.4%  of  the  animals  in  infected  herds 
which  have  no  history  of  abortion,  show  evidence  of  infection 
with  abortion  bacilli.  This  infection  may  have  occurred  either 
while  the  animal  was  in  utero  (congenital  immunity)  or  have 
been  acquired  after  birth.  These  figures,  of  course,  include  a 


248 


Wisconsin  Research  Bulletin  No.  24 


certain  number  of  cows  that  have  been  bought  from  unknown 
sources  and  which,  if  the  actual  history  were  known,  may  have 
aborted  in  the  past.  Only  83.5%  of  the  cows  which  have  been 
known  to  abort  gave  positive  reactions.  This  apparent  discre- 
pancy is  explained  when  attention  is  drawn  to  the  fact  that  many 
of  these  animals  aborted  more  than  a year  before  the  test  was 
applied,  while  a few  aborted  four  or  five  years  before.  It  is 
assumed  that  the  negative  reactors  in  this  group  have-  lost  what- 
ever immunity  they  may  have  gained  from  the  infection.  Only 
one  herd  bull  out  of  ten  ga  ve  a positive  reaction,  indicating  that 
the  male  in  these  cases  rarely  harbored  the  abortion  bacilli  in  suf- 
ficient quantities  to  stimulate  the  formation  of  immune  bodies 
m his  serum.  Of  the  entire  411  animals  tested  135,  or  32.9% 
gave  positive  reactions  and  must,  therefore,  be  considered  as  har- 
boring the  abortion  bacilli  at  the  time  the  test  was  made,  or  at 
some  time  in  the  not  distant  past.  Two  hundred  and  sixty-one 
or  63.5%,  were  found  free  from  the  infection.  Of  these,  four- 
teen, had  been  known  to  abort;  some,  from  accidental  causes, 
others,  possibly  from  the  contagious  form,  but  no  immune  bodies 
could  be  demonstrated  in  their  sera.  It  must  be  understood 
that  practically  all  these  animals  were  in  infected  herds,  in  fact, 
97  were  known  to  have  aborted;  therefore,  32.9%  is  not  a larger 
number  of  reactors  than  should  reasonably  be  expected.  No  con. 
elusions  as  to  the  prevalency  of  the  disease  can  be  gathered  from 
these  figures  because  the  average  dairy  herd  is  not  represented, 
instead  only  those  herds  have  been  included  in  which  the  infec- 
tion  was  known  to  exist. 


RESEARCH  BULLETIN  NO.  24 


JUNE,  1912 


THE  UNIVERSITY  OF  WISCONSIN 
AGRICULTURAL  EXPERIMENT  STATION 


The  Diagnosis  of  Contagious  Abortion  in 
Cattle  by  Means  of  the  Complement 
Fixation  Test 

V i ■ BY  ~ 

F.  B.  HADLEY  and  B.  A.  BEACH 


MADISON,  WISCONSIN 


A 


**  - 


UNIVERSITY  OF  ILLINOIS-URBANA 
con  7U/75RE  CO 02 

RESEARCH  BULLETIN  MADISON 
14-24  1911-12 


019935896 


